Compounds and methods for modulating claudin-mediated functions

ABSTRACT

Methods for using modulating agents to enhance or inhibit claudin-mediated cell adhesion in a variety of in vivo and in vitro contexts are provided. Within certain embodiments, the modulating agents may be used to increase blood/brain barrier permeability. The modulating agents comprise at least one claudin cell adhesion recognition sequence or an antibody or fragment thereof that specifically binds the claudin cell adhesion recognition sequence. Modulating agents may additionally comprise one or more cell adhesion recognition sequences recognized by other adhesion molecules. Such modulating agents may, but need not, be linked to a targeting agent, drug and/or support material.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. application Ser. No.09/282,029, filed Mar. 30, 1999 now U.S. Pat. No. 6,723,700, which is acontinuation-in-part of U.S. application Ser. No. 09/185,908, filed Nov.3, 1998 now U.S. Pat. No. 6,756,356.

TECHNICAL FIELD

The present invention relates generally to methods for regulatingclaudin-mediated processes, and more particularly to the use ofmodulating agents comprising a claudin cell adhesion recognitionsequence and/or an antibody that specifically recognizes such a sequencefor inhibiting functions such as cell adhesion and the formation oftissue permeability barriers.

BACKGROUND OF THE INVENTION

Cell adhesion is a complex process that is important for maintainingtissue integrity and generating physical and permeability barrierswithin the body. All tissues are divided into discrete compartments,each of which is composed of a specific cell type that adheres tosimilar cell types. Such adhesion triggers the formation ofintercellular junctions (i.e., readily definable contact sites on thesurfaces of adjacent cells that are adhering to one another), also knownas tight junctions, gap junctions, spot desmosomes and belt desmosomes.The formation of such junctions gives rise to physical and permeabilitybarriers that restrict the free passage of cells and other biologicalsubstances from one tissue compartment to another. For example, theblood vessels of all tissues are composed of endothelial cells. In orderfor components in the blood to enter a given tissue compartment, theymust first pass from the lumen of a blood vessel through the barrierformed by the endothelial cells of that vessel. Similarly, in order forsubstances to enter the body via the gut, the substances must first passthrough a barrier formed by the epithelial cells of that tissue. Toenter the blood via the skin, both epithelial and endothelial celllayers must be crossed.

Cell adhesion is mediated by specific cell surface adhesion molecules(CAMs). There are many different families of CAMs, including theimmunoglobulin, integrin, selectin and cadherin superfamilies, and eachcell type expresses a unique combination of these molecules. Cadherinsare a rapidly expanding family of calcium-dependent CAMs (Munro et al.,In: Cell Adhesion and Invasion in Cancer Metastasis, P. Brodt, ed., pp.17-34, RG Landes Co., Austin Tex., 1996). The cadherins (abbreviatedCADs) are membrane glycoproteins that generally promote cell adhesionthrough homophilic interactions (a CAD on the surface of one cell bindsto an identical CAD on the surface of another cell). Cadherins have beenshown to regulate epithelial, endothelial, neural and cancer celladhesion, with different CADs expressed on different cell types. Forexample, N (neural)—cadherin is predominantly expressed by neural cells,endothelial cells and a variety of cancer cell types. E(epithelial)—cadherin is predominantly expressed by epithelial cells. VE(vascular endothelial)—cadherin is predominantly expressed byendothelial cells. Other CADs are P (placental)—cadherin, which is foundin human skin, and R (retinal)—cadherin. A detailed discussion of thecadherins is provided in Munro S B et al., 1996, In: Cell Adhesion andInvasion in Cancer Metastasis, P. Brodt, ed., pp.17-34 (RG LandesCompany, Austin Tex.) and Lampugnani and Dejana, Curr. Opin. Cell Biol.9:674-682, 1997.

CAD-mediated cell adhesion triggers a cascade of events that lead to theformation of intercellular junctions, and ultimately to theestablishment of permeability barriers between tissue compartments. Theintercellular junction that is directly responsible for the creation ofpermeability barriers that prevent the diffusion of solutes throughparacellular spaces is known as the tight junction, or zonula occludens(Anderson and van Itallie, Am. J. Physiol. 269:G467-G475, 1995;Lampugnani and Dejana, Curr. Opin. Cell Biol. 9:674-682, 1997).

The transmembrane component of tight junctions that has been the moststudied is occludin (Furuse et al., J. Cell Biol. 123:1777-1788, 1993;Furuse et al., J. Cell Sci. 109:429-435, 1996). This protein appears tobe expressed by all endothelial cell types, as well as by mostepithelial cell types. Occludin is an integral membrane protein that iscomposed of two extracellular domains, four hydrophobic domains thattransverse the plasma membrane, and three cytoplasmic domains, and thestructures of all known mammalian occludins are similar (Ando-Akatsukaet al., J. Biol. Chem. 133:43-47, 1996). Occludin is believed to bedirectly involved in cell adhesion and the formation of tight junctions(Furuse et. al., J. Cell Sci. 109:429-435, 1996, Chen et al., J. CellBiol. 138:891-899, 1997). It has been proposed that occludin promotescell adhesion through homophilic interactions (an occludin on thesurface of one cell binds to an identical occludin on the surface ofanother cell). A detailed discussion of occludin structure and functionis provided by Lampugnani and Dejana, Curr. Opin. Cell Biol. 9:674-682,1997.

More recently, a second family of tight junction components has beenidentified. Claudins are transmembrane proteins that appear to bedirectly involved in cell adhesion and the formation of tight junctions(Furuse et al., J. Cell Biology 141:1539-1550, 1998; Morita et al.,Proc. Natl. Acad Sci. USA 96:511-516, 1999). Other previously describedproteins that appear to be members of the claudin family include RVP-1(Briehl and Miesfeld, Molecular Enidocrinology 5:1381-1388, 1991;Katahira et al., J. Biological Chemistry 272:26652-26656, 1997), theClostridium perfringens enterotoxin receptor (CPE-R; see Katahira etal., i J. Cell Biology 136:1239-1247, 1997; Katahira et al., J.Biological Chemistry 272:26652-26656, 1997) and TMVCF (transmembraneprotein deleted in Velo-cardio-facial syndrome; Sirotkin et al.,Genomics 42:245-51, 1997).

Based on hydrophobicity analysis, all claudins appear to beapproximately 22 kD and contain four hydrophobic domains that transversethe plasma membrane. It has been proposed that claudins promote celladhesion through homophilic interactions (a claudin on the surface ofone cell binds to an identical claudin on the surface of another cell)or heterophilic interactions, possibly with occludin.

Although cell adhesion is required for certain normal physiologicalfunctions, there are situations in which the level of cell adhesion isundesirable. For example, many pathologies (such as autoimmune diseasesand inflammatory diseases) involve abnormal cellular adhesion. Celladhesion may also play a role in graft rejection. In such circumstances,modulation of cell adhesion may be desirable.

In addition, permeability barriers arising from cell adhesion createdifficulties for the delivery of drugs to specific tissues and tumorswithin the body. For example, skin patches are a convenient tool foradministering drugs through the skin. However, the use of skin patcheshas been limited to small, hydrophobic molecules because of theepithelial and endothelial cell barriers. Similarly, endothelial cellsrender the blood capillaries largely impermeable to drugs, and theblood/brain barrier has hampered the targeting of drugs to the centralnervous system. In addition, many solid tumors develop internal barriersthat limit the delivery of anti-tumor drugs and antibodies to innercells.

Attempts to facilitate the passage of drugs across such barriersgenerally rely on specific receptors or carrier proteins that transportmolecules across barriers in vivo. However, such methods are ofteninefficient, due to low endogenous transport rates or to the poorfunctioning of a carrier protein with drugs. While improved efficiencyhas been achieved using a variety of chemical agents that disrupt celladhesion, such agents are typically associated with undesirableside-effects, may require invasive procedures for administration and mayresult in irreversible effects.

Accordingly, there is a need in the art for compounds that modulate celladhesion and improve drug delivery across permeability barriers withoutsuch disadvantages. The present invention fulfills this need and furtherprovides other related advantages.

SUMMARY OF THE INVENTION

The present invention provides compounds and methods for modulatingclaudin-mediated cell adhesion and the formation of permeabilitybarriers. Within certain aspects, the present invention provides celladhesion modulating agents that inhibit or enhance claudin-mediated celladhesion. Certain modulating agents (a) comprise a claudin CAR sequence;and (b) contain 3-16 amino acid residues linked by peptide bonds. Othermodulating agents (a) comprise at least five or seven consecutive aminoacid residues of a claudin CAR sequence having the formula:

Trp-Lys/Arg/Gln-Aaa-Baa-Ser/Ala-Tyr/Phe-Caa-Gly/Asp/Glu (SEQ ID NO:1)

wherein Aaa, Baa and Caa indicate independently selected amino acidresidues; Lys/Arg/Gln is an amino acid that is lysine, arginine orglutamine, Ser/Ala is an amino acid that is serine or alanine; Tyr/Pheis an amino acid that is tyrosine or phenylalanine; and Gly/Asp/Glu isan amino acid that is glycine, aspartic acid or glutamic acid; and (b)contain no more than 50 consecutive amino acid residues present withinthe claudin. Still other modulating agents (a) comprise at least eightconsecutive amino acid residues of a claudin CAR sequence having theabove formula; and (b) contain no more than 50 consecutive amino acidresidues present within the claudin.

Within certain embodiments, a modulating agent as described abovecomprises a claudin CAR sequence that is present within a cyclicpeptide. The cyclic peptide may have the formula:

wherein W is a tetrapeptide selected from the group consisting of IYSY(SEQ ID NO:2), TSSY (SEQ ID NO:3), VTAF (SEQ ID NO:4), VSAF (SEQ IDNO:5) and MSSY (SEQ ID NO:386); wherein X₁, and X₂ are optional, and ifpresent, are independently selected from the group consisting of aminoacid residues and combinations thereof in which the residues are linkedby peptide bonds, and wherein X₁ and X₂ independently range in size from0 to 10 residues, such that the sum of residues contained within X₁ andX₂ ranges from 1 to 12; wherein Y₁ and Y₂ are independently selectedfrom the group consisting of amino acid residues, and wherein a covalentbond is formed between residues Y₁ and Y₂; and wherein Z₁ and Z₂ areoptional, and if present, are independently selected from the groupconsisting of amino acid residues and combinations thereof in which theresidues are linked by peptide bonds. In certain embodiments, Y₁comprises an N-acetyl group and/or Y₂ comprises a C-terminal amidegroup. Y₁ and Y₂ may be covalently linked via any suitable bond,including a disulfide bond, an amide bond or a thioether bond.

The present invention further provides, within other aspects,polynucleotides encoding a modulating agent as provided above,expression vectors comprising such a polynucleotide, and host cellstransformed or transfected with such an expression vector.

Within further aspects, the present invention provides modulating agentsthat comprise an antibody or antigen-binding fragment thereof thatspecifically binds to a claudin CAR sequence and modulates aclaudin-mediated function, wherein the claudin CAR sequence has theformula:

Trp-Lys/Arg/Gln-Aaa-Baa-Ser/Ala-Tyr/Phe-Caa-Gly/Asp/Glu (SEQ ID NO:1)

wherein Aaa, Baa and Caa indicate independently selected amino acidresidues; Lys/Arg/Gln is an amino acid that is lysine, arginine orglutamine; Ser/Ala is an amino acid that is serine or alanine; Tyr/Pheis an amino acid that is tyrosine or phenylalanine; and Gly/Asp/Glu isan amino acid that is glycine, aspartic acid or glutamic acid.

The present invention further provides modulating agents comprising amimetic of a claudin CAR sequence that comprises at least three or fiveconsecutive amino acid residues of a claudin CAR sequence having theformula

Trp-Lys/Arg/Gln-Aaa-Baa-Ser/Ala-Tyr/Phe-Caa-Gly/Asp/Glu (SEQ ID NO:1)

wherein Aaa, Baa and Caa indicate independently selected amino acidresidues; Lys/Arg/Gln is an amino acid that is lysine, arginine orglutamine; Ser/Ala is an amino acid that is serine or alanine; Tyr/Pheis an amino acid that is tyrosine or phenylalanine; and Gly/Asp/Glu isan amino acid that is glycine, aspartic acid or glutamic acid; andwherein the mimetic is capable of modulating a claudin-mediatedfunction.

Within other aspects, modulating agents as described above may be linkedto one or more of a drug, a detectable marker, a targeting agent and/ora support material. Alternatively, or in addition, modulating agents asdescribed above may further comprise one or more of: (a) a cell adhesionrecognition sequence that is bound by an adhesion molecule other than aclaudin, wherein the cell adhesion recognition sequence is separatedfrom any claudin CAR sequence(s) by a linker; and/or (b) an antibody orantigen-binding fragment thereof that specifically binds to a celladhesion recognition sequence bound by an adhesion molecule other than aclaudin. Such adhesion molecules may be selected from the groupconsisting of integrins, cadherins, occludin, N-CAM, JAM, PE-CAM,desmogleins, desmocollins, fibronectin, laminin and other extracellularmatrix proteins.

Within certain specific aspects, a modulating agent may comprise one ormore claudin-1 CAR sequences selected from the group consisting of: IYSY(SEQ ID NO:2), IYSYA (SEQ ID NO:27), IYSYAG (SEQ ID NO:28), KIYSY (SEQID NO:29), KIYSYA (SEQ ID NO:30), KIYSYAG (SEQ ID NO:31), WKIYSY (SEQ IDNO:32), WKIYSYA (SEQ ID NO:33), WKIYSYAG (SEQ ID NO:34), WKIYSYAGN (SEQID NO:476), RIYSY (SEQ ID NO:480), RIYSYA (SEQ ID NO:481), RIYSYAG (SEQID NO:482), WRIYSY (SEQ ID NO:483), WRIYSYA (SEQ ID NO:484), WRIYSYAG(SEQ ID NO:485), and WRIYSYAGN (SEQ ID NO:486). Such CAR sequences maybe present within a cyclic peptide, such as: CIYSYC (SEQ ID NO:59),CIYSYAC (SEQ ID NO:60), CIYSYAGC (SEQ ID NO:61), CKIYSYC (SEQ ID NO:62),CKIYSYAGC (SEQ ID NO:63), CKIYSYAGC (SEQ ID NO:64), CWKIYSYC (SEQ IDNO:65), CWKIYSYAC (SEQ ID NO:66), CWKIYSYAGC (SEQ ID NO:67), CRIYSYC(SEQ ID NO:487), CRIYSYAC (SEQ ID NO:488), CRIYSYAGC (SEQ ID NO:489),CWRIYSYC (SEQ ID NO:490), CWRIYSYAC (SEQ ID NO:491), CWRIYSYAGC (SEQ IDNO:492), KIYSYD (SEQ ID NO:68), KIYSYAD (SEQ ID NO:69), KIYSYAGD (SEQ IDNO:70), KKIYSYD (SEQ ID NO:71), KKIYSYAD (SEQ ID NO:72), KKIYSYAGD (SEQID NO:73), KWKIYSYD (SEQ ID NO:74), KWKIYSYAD (SEQ ID NO:75), KWKIYSYAGD(SEQ ID NO:76), KRIYSYD (SEQ ID NO:493), KRIYSYAD (SEQ ID NO:494),KRIYSYAGD (SEQ ID NO:495), KWRIYSYD (SEQ ID NO:496), KWRIYSYAD (SEQ IDNO:497), KWRIYSYAGD (SEQ ID NO:498), KIYSYED (SEQ ID NO:77), KIYSYAE(SEQ ID NO:78), KIYSYAGE (SEQ ID NO:79), KKIYSYE (SEQ ID NO:80),KKIYSYAE (SEQ ID NO:81), KKIYSYAGE (SEQ ID NO:82), KWKIYSYE (SEQ IDNO:83), KWKIYSYAE (SEQ ID NO:84), KWKIYSYAGE (SEQ ID NO:85), KRIYSYE(SEQ ID NO:499), KRIYSYAE (SEQ ID NO:500), KRIYSYAGE (SEQ ID NO:501),KWRIYSYE (SEQ ID NO:502), KWRIYSYAE (SEQ ID NO:503), KWRIYSYAGE (SEQ IDNO:504), DIYSYK (SEQ ID NO:86), DIYSYAK (SEQ ID NO:87), DIYSYAGK (SEQ IDNO:88), DKIYSYK (SEQ ID NO:89), DKIYSYAK (SEQ ID NO:90), DKIYSYAGK (SEQID NO:91), DWKISYK (SEQ ID NO:92), DWKISYAK (SEQ ID NO:93), DWKISYAGK(SEQ ID NO:94), DRIYSYK (SEQ ID NO:505), DRIYSYAK (SEQ ID NO:506),DRIYSYAGK (SEQ ID NO:507), DWRIYSYK (SEQ ID NO:508), DWRIYSYAK (SEQ IDNO:509), DWRIYSYAGK (SEQ ID NO:510), EIYSYK (SEQ ID NO:95), EIYSYAK (SEQID NO:96), EIYSYAGK (SEQ ID NO:97), EKIYSYK (SEQ ID NO:98), EKIYSYAK(SEQ ID NO:99), EKIYSYAGK (SEQ ID NO:100), EWKIYSYK (SEQ ID NO:101),EWKIYSYAK (SEQ ID NO:102), EWKIYSYAGK (SEQ ID NO:103), ERIYSYK (SEQ IDNO:511), ERIYSYAK (SEQ ID NO:512), ERIYSYAGK (SEQ ID NO:513), EWRIYSYK(SEQ ID NO:514), EWRIYSYAK (SEQ ID NO:515), EWRIYSYAGK (SEQ ID NO:516),IYSYA (SEQ ID NO:104), IYSYAG (SEQ ID NO:105), KIYSY (SEQ ID NO:106),KIYSYAG (SEQ ID NO:107), WKIYSY (SEQ ID NO:108), WKIYSYA (SEQ IDNO:109), WKIYSYAG (SEQ ID NO:110), WRIYSY (SEQ ID NO:517), WRIYSYA (SEQID NO:518) or WRIYSYAG (SEQ ID NO:519).

Within other aspects, a modulating agent may comprise an antibody orantigen-binding fragment thereof that specifically binds to theclaudin-1 CAR sequence WKIYSYAG (SEQ ID NO:34) or WRIYSYAG (SEQ IDNO:485).

Within further aspects, a modulating agent as described above maycomprise one or more claudin-2 CAR sequences selected from the groupconsisting of: TSSY (SEQ ID NO:3), TSSYV (SEQ ID NO:35), TSSYVG (SEQ IDNO:36), RTSSY (SEQ ID NO:37), RTSSYV (SEQ ID NO:38), RTSSYVG (SEQ IDNO:39), WRTSSY (SEQ ID NO:40), WRTSSYV (SEQ ID NO:41) and WRTSSYVG (SEQID NO:42). Such CAR sequences may be present within a cyclic peptide,such as: CTSSYC (SEQ ID NO 111), CTSSYVC (SEQ ID NO:112), CTSSYVGC (SEQID NO:113), CRTSSY (SEQ ID NO:114), CRTSSYVC (SEQ ID NO:115), CRTSSYVGC(SEQ ID NO:116), CWRTSSYC (SEQ ID NO:117), CWRTSSYVC (SEQ ID NO:118),CWRTSSYVGC (SEQ ID NO:119), KTSSYD (SEQ ID NO:120), KTSSYVD (SEQ IDNO:121), KTSSYVGD (SEQ ID NO:122), KRTSSYD (SEQ ID NO:123), KRTSSYVD(SEQ ID NO:124), KRTSSYVGD (SEQ ID NO:125), KWRTSSYD (SEQ ID NO:126),KWRTSSYVD (SEQ ID NO:127), KWRTSSYVGD (SEQ ID NO:128), KTSSYE (SEQ IDNO:129), KTSSYVE (SEQ ID NO:130), KTSSYVGE (SEQ ID NO:131), KRTSSYE (SEQID NO:132), KRTSSYVE:(SEQ ID NO:133), KRTSSYVGE (SEQ ID NO:134),KWRTSSYE (SEQ ID NO:135), KWRTSSYVE (SEQ ID NO:136), KWRTSSYVGE (SEQ IDNO:137), DTSSYK (SEQ ID NO:138), DTSSYVK (SEQ ID NO:139), DTSSYVGK (SEQID NO:140), DRTSSYK (SEQ ID NO:141), DRTSSYVK (SEQ ID NO:142), DRTSSYVGK(SEQ ID NO:143), DWRTSSYK (SEQ ID NO:144), DWRTSSYVK (SEQ ID NO:145),DWRTSSYVGK (SEQ ID NO:146), ETSSYK (SEQ ID NO:147), ETSSYVK (SEQ IDNO:148), ETSSYVGK (SEQ ID NO:149), ERTSSYK (SEQ ID NO:150), ERTSSYVK(SEQ ID NO:151), ERTSSYVGK (SEQ ID NO:152), EWRTSSYK (SEQ ID NO:153),EWRTSSYVK (SEQ ID NO:154), EWRTSSYVGK (SEQ ID NO:155), TSSYV (SEQ IDNO:156), TSSYVG (SEQ ID NO:157), RTSSY (SEQ ID NO:158), RTSSYV (SEQ IDNO:159), RTSSYVG (SEQ ID NO:160), WRTSSY (SEQ ID NO:161), WRTSSYV (SEQID NO:162) and WRTSSYVG (SEQ ID NO:163).

Within other aspects, a modulating agent may comprise an antibody orantigen-binding fragment thereof that specifically binds to theclaudin-2 CAR sequence WRTSSYVG (SEQ ID NO:42).

Within further aspects, a modulating agent comprises one or moreclaudin-3 CAR sequences selected from the group consisting of: VSAF (SEQID NO:5), VSAFI (SEQ ID NO:51), VSAFIG (SEQ ID NO:52), RVSAF (SEQ IDNO:53), RVSAFI (SEQ ID NO:54), RVSAFIG (SEQ ID NO:55), WRVSAF (SEQ IDNO:56), WRVSAFI (SEQ ID NO:57) and WRVSAFIG (SEQ ID NO:58). Such CARsequences may be present within a cyclic peptide, such as: CVSAFC (SEQID NO:217), CVSAFIC (SEQ ID NO:218), CVSAFIGC (SEQ ID NO:219), CRVSAFC(SEQ ID NO:220), CRVSAFIC (SEQ ID NO:221), CRVSAFIGC (SEQ ID NO:222),CWRVSAFC (SEQ ID NO:223), CWRVSAFIC (SEQ ID NO:224), CWRVSAFIGC (SEQ IDNO:225), KVSAFD (SEQ ID NO:226), KVSAFID (SEQ ID NO:227), KVSAFIGD (SEQID NO:228), KRVSAFD (SEQ ID NO:229), KRVSAFID (SEQ ID NO:230), KRVSAFIGD(SEQ ID NO:231), KWRVSAFD (SEQ ID NO:232), KWRVSAFID (SEQ ID NO:233),KWRVSAFIGD (SEQ ID NO:234), KVSAFE (SEQ ID NO:235), KVSAFIE (SEQ IDNO:236), KVSAFIGE (SEQ ID NO:237), KRVSAFE (SEQ ID NO:238), KRVSAFIE(SEQ ID NO:239), KRVSAFIGE (SEQ ID NO:240), KWRVSAFE (SEQ ID NO:241),KWRVSAFIE (SEQ ID NO:242), KWRVSAFIGE (SEQ ID NO:243), DVSAFK (SEQ IDNO:244), DVSAFIK (SEQ ID NO:245), DVSAFIGK (SEQ ID NO:246), DRVSAFK (SEQID NO:247), DRVSAFIK (SEQ ID NO:248), DRVSAFIGK (SEQ ID NO:249),DWRVSAFK (SEQ ID NO:250), DWRVSAFIK (SEQ ID NO:251), DWRVSAFIGK (SEQ IDNO:252), EVSAFK (SEQ ID NO:253), EVSAFIK (SEQ ID NO:254), EVSAFIGK (SEQID NO:255), ERVSAFK (SEQ ID NO:256), ERVSAFIK (SEQ ID NO:257), ERVSAFIGK(SEQ ID NO:258), EWRVSAFK (SEQ ID NO:259), EWRVSAFIK (SEQ ID NO:260),EWRVSAFIGK (SEQ ID NO:261), VSAFI (SEQ ID NO:262), VSAFIG (SEQ IDNO:263), RVSAF (SEQ ID NO:264), RVSAFI (SEQ ID NO:265), RVSAFIG (SEQ IDNO:266), WRVSAF (SEQ ID NO:267), WRVSAFI (SEQ ID NO:268) and WRVSAFIG(SEQ ID NO:269).

Within other aspects, a modulating agent may comprise an antibody orantigen-binding fragment thereof that specifically binds to theclaudin-3 CAR sequence WRVSAFIG (SEQ ID NO:58).

Modulating agents may comprise, within other aspects one or moreclaudin-4 CAR sequences selected from the group consisting of: VTAF (SEQID NO:4), VTAFI (SEQ ID NO:43), VTAFIG (SEQ ID NO:44): RVTAF (SEQ IDNO:45), RVTAFI (SEQ ID NO:46), RVTAFIG (SEQ ID NO:47), WRVTAF (SEQ IDNO:48), WRVTAFI (SEQ ID NO:49) and WRVTAFIG (SEQ ID NO:50). Such CARsequences may be present within a cyclic peptide, such as: CVTAFC (SEQID NO:164), CVTAFIC (SEQ ID NO:165), CVTAFIGC (SEQ ID NO:166),CRVTAFC(SEQ ID NO:167), CRVTAFIC (SEQ ID NO:168), CRVTAFIGC (SEQ IDNO:169), CWRVTAFC (SEQ ID NO:170), CWRVTAFIC (SEQ ID NO:171), CWRVTAFIGC(SEQ ID NO: 172), KVTAFD (SEQ ID NO: 173), KVTAFID (SEQ ID NO: 174),KVTAFIGD (SEQ ID NO:175), KRVTAFD (SEQ ID NO: 176), KRVTAFID (SEQ IDNO:177), KRVTAFIGD (SEQ ID NO:178), KWRVTAFD (SEQ ID NO:179), KWRVTAFID(SEQ ID NO:180), KWRVTAFIGD (SEQ ID NO:181), KVTAFE (SEQ ID NO:182),KVTAFIE (SEQ ID NO:183), KVTAFIGE (SEQ ID NO:184), KRVTAFE (SEQ IDNO:185), KRVTAFIE (SEQ ID NO:186), KRVTAFIGE (SEQ ID NO:187), KWRVTAFE(SEQ ID NO:188), KWRVTAFIE (SEQ ID NO:189), KWRVTAFIGE (SEQ ID NO: 190),DVATFK (SEQ ID NO: 191), DVTAFIK (SEQ ID NO:192), DVTAFIGK (SEQ IDNO:193), DRVTAFK (SEQ ID NO:194), DRVTAFIK (SEQ ID NO: 195), DRVTAFIGK(SEQ ID NO: 196), DWRVTAFK (SEQ ID NO:197), DWRVTAFIK (SEQ ID NO:198),DWRVTAFIGK (SEQ ID NO:199), EVTAFK (SEQ ID NO:200), EVTAFIK (SEQ IDNO:201), EVTAFIGK (SEQ ID NO:202), ERVTAFK (SEQ ID NO:203), ERVITAFIK(SEQ ID NO:204), ERVTAFIGK (SEQ ID NO:205), EWRVTAFK (SEQ ID NO:206),EWRVTAFIK (SEQ ID NO:207), EWRVTAFIGK (SEQ ID NO:208), VTAFI (SEQ IDNO:209), VTAFIG (SEQ ID NO:210), RVTAF (SEQ ID NO:211), RVTAFI (SEQ IDNO:212), RVTAFIG (SEQ ID NO:213), WRVTAF (SEQ ID NO:214), WRVTAFI (SEQID NO:215) and WRVTAFIG (SEQ ID NO:216).

Within other aspects, a modulating agent may comprise an antibody orantigen-binding fragment thereof that specifically binds to theclaudin-4 CAR sequence WRVTAFIG (SEQ ID NO:50).

Modulating agents may comprise, within other aspects one or moreclaudin-5 CAR sequences selected from the group consisting of: VTAF (SEQID NO:4), VTAFL (SEQ ID NO:270), VTAFLD (SEQ ID NO:271), QVTAF (SEQ IDNO:272), QVTAFL (SEQ ID NO:273), QVTAFLD (SEQ ID NO:274), WQVTAF (SEQ IDNO:275), WQVTAFL (SEQ ID NO:276) and WQVTAFLD (SEQ ID NO:277). Such CARsequences may be present within a cyclic peptide, such as: CVTAFC (SEQID NO:164), CVTAFLC (SEQ ID NO:278), CVTAFLDC (SEQ ID NO:279), CQVTAFC(SEQ ID NO:280), CQVTAFLC (SEQ ID NO:281), CQVTAFLDC (SEQ ID NO:282),CWQVTAFC (SEQ ID NO:283), CWQVTAFLC (SEQ ID NO:284), CWQVTAFLDC (SEQ IDNO:285), KVTAFD (SEQ ID NO:286), KVTAFLD (SEQ ID NO:287), KVTAFLDD (SEQID NO:288), KQVTAFD (SEQ ID NO:289), KQVTAFLD (SEQ ID NO:290), KQVTAFLDD(SEQ ID NO:291), KWQVTAFD (SEQ ID NO:292), KWQVTAFLD (SEQ ID NO:293),KWQVTAFLDD (SEQ ID NO:294), KVTAFE (SEQ ID NO:182), KVTAFLE (SEQ IDNO:295), KVTAFLDE (SEQ ID NO:296), KQVTAFE (SEQ ID NO:297), KQVTAFLE(SEQ ID NO:298), KQVTAFLDE (SEQ ID NO:299), KWQVTAFE (SEQ ID NO:300),KWQVTAFLE (SEQ ID NO:301), KWQVTAFLDE (SEQ ID NO:302), DVATFK (SEQ IDNO:303), DVTAFLK (SEQ ID NO:304), DVTAFLDK (SEQ ID NO:305), DQVTAFK (SEQID NO:306), DQVTAFLK (SEQ ID NO:307), DQVTAFLDK (SEQ ID NO:308),DWQVTAFK (SEQ ID NO:309), DWQVTAFLK (SEQ ID NO:310), DWQVTAFLDK (SEQ IDNO:311), EVTAFK (SEQ ID NO:200), EVTAFLK (SEQ ID NO:312), EVTAFLDK (SEQID NO:313), EQVTAFK (SEQ ID NO:314), EQVTAFLK (SEQ ID NO:315), EQVTAFLDK(SEQ ID NO: 316), EWQVTAFK (SEQ ID NO:317), EWQVTAFLK (SEQ ID NO:318),EWQVTAFLDK (SEQ ID NO:319), VTAFL (SEQ ID NO:320), VTAFLD (SEQ IDNO:321), QVTAF (SEQ ID NO:322), QVTAFL (SEQ ID NO:323), QVTAFLD (SEQ IDNO:324), WQVTAF (SEQ ID NO:325), WQVTAFL (SEQ ID NO:326) and WQVTAFLD(SEQ ID NO:327).

Within other aspects, a modulating agent may comprise an antibody orantigen-binding fragment thereof that specifically binds to theclaudin-5 CAR sequence WQVTAFLD (SEQ ID NO:277).

Modulating agents may comprise, within other aspects one or moreclaudin-6 or claudin-9 CAR sequences selected from the group consistingof: VTAF (SEQ ID NO:4), VTAFI (SEQ ID NO:328), VTAFIG (SEQ ID NO:329),KVTAF (SEQ ID NO:330), KVTAFI (SEQ ID NO:331), KVTAFIG (SEQ ID NO:332),WKVTAF (SEQ ID NO:333), WKVTAFI (SEQ ID NO:334) and WKVTAFIG (SEQ IDNO:335). Such CAR sequences may be present within a cyclic peptide, suchas: CVTAFC (SEQ ID NO:164), CVTAFIC (SEQ ID NO:336), CVTAFIGC (SEQ IDNO:337), CKVTAFC (SEQ ID NO:338), CKVTAFIC (SEQ ID NO:339), CKVTAFIGC(SEQ ID NO:340), CWKVTAFC (SEQ ID NO:341), CWKVTAFIC (SEQ ID NO:342),CWKVTAFIGC (SEQ ID NO:343), KVTAFD (SEQ ID NO:344), KVTAFID (SEQ IDNO:345), KVTAFIGD (SEQ ID NO:346), KKVTAFD (SEQ ID NO:347), KKVTAFID(SEQ ID NO:348), KKVTAFIGD (SEQ ID NO:349), KWKVTAFD (SEQ ID NO:350),KWKVTAFID (SEQ ID NO:351), KWKVTAFIGD (SEQ ID NO:352), KVTAFE (SEQ IDNO:182), KVTAFIE (SEQ ID NO:353), KVTAFIGE (SEQ ID NO:354), KKVTAFE (SEQID NO:355), KKVTAFIE (SEQ ID NO:356), KKVTAFIGE (SEQ ID NO:357),KWKVTAFE (SEQ ID NO:358), KWKVTAFIE (SEQ ID NO:359), KWKVTAFIGE (SEQ IDNO:360), DVATFK (SEQ ID NO:361), DVTAFIK (SEQ ID NO:362), DVTAFIGK (SEQID NO:363), DKVTAFK (SEQ ID NO:364), DKVTAFIK (SEQ ID NO:365), DKVTAFIGK(SEQ ID NO:366), DWKVTAFK (SEQ ID NO:367), DWKVTAFIK (SEQ ID NO:368),DWKVTAFIGK (SEQ ID NO:369), EVTAFK (SEQ ID NO:200), EVTAFIK (SEQ IDNO:370), EVTAFIGK (SEQ ID NO:371), EKVTAFK (SEQ ID NO:372), EKVTAFIK(SEQ ID NO:373), EKVTAFIGK (SEQ ID NO:374), EWKVTAFK (SEQ ID NO:375),EWKVTAFIK (SEQ ID NO:376), EWKVTAFIGK (SEQ ID NO:377), VTAFI (SEQ IDNO:378), VTAFIG (SEQ ID NO:379), KVTAF (SEQ ID NO:380), KVTAFI (SEQ IDNO:381), VTAFIG (SEQ ID NO:382), WKVTAF (SEQ ID NO:383), WKVTAFI (SEQ IDNO:384) and WKVTAFIG (SEQ ID NO:385).

Within other aspects, a modulating agent may comprise an antibody orantigen-binding fragment thereof that specifically binds to theclaudin-6 and claudin-9 CAR sequence WKVTAFIG (SEQ ID NO:335).

Within further aspects, a modulating agent as described above maycomprise one or more claudin-7 CAR sequences selected from the groupconsisting of: MSSY (SEQ ID NO:386), MSSYA (SEQ ID NO:387), MSSYAG (SEQID NO:388), QMSSY (SEQ ID NO:389), QMSSYA (SEQ ID NO:390), QMSSYAG (SEQID NO:391), WQMSSY (SEQ ID NO:392), WQMSSYA (SEQ ID NO:393) and WQMSSYAG(SEQ ID NO:394). Such CAR sequences may be present within a cyclicpeptide, such as: CMSSYC (SEQ ID NO:395), CMSSYAC (SEQ ID NO:396),CMSSYAGC (SEQ ID NO:397), CQMSSYC (SEQ ID NO:398), CQMSSYAC (SEQ IDNO:399), CQMSSYAGC (SEQ ID NO:400), CWQMSSYC (SEQ ID NO:401), CWQMSSYAC(SEQ ID NO:402), CWQMSSYAGC (SEQ ID NO:403), KMSSYD (SEQ ID NO:404),KMSSYAD (SEQ ID NO:405), KMSSYAGD (SEQ ID NO:406), KQMSSYD (SEQ IDNO:407), KQMSSYAD (SEQ ID NO:408), KQMSSYAGD (SEQ ID NO:409), KWQMSSYD(SEQ ID NO:410), KWQMSSYAD (SEQ ID NO:411), KWQMSSYAGD (SEQ ID NO:412),KMSSYE (SEQ ID NO:413), KMSSYAE (SEQ ID NO:414), KMSSYAGE (SEQ IDNO:415), KQMSSYE (SEQ ID NO:416), KQMSSYAE (SEQ ID NO:417), KQMSSYAGE(SEQ ID NO:418), KWQMSSYE (SEQ ID NO:419), KWQMSSYAE (SEQ ID NO:420),KWQMSSYAGE (SEQ ID NO:421), DMSSYK (SEQ ID NO:422), DMSSYAK (SEQ IDNO:423), DMSSYAGK (SEQ ID NO:424), DQMSSYK (SEQ ID NO:425), DQMSSYAK(SEQ ID NO:426), DQMSSYAGK (SEQ ID NO:427), DWQMSSYK (SEQ ID NO:428),DWQMSSYAK (SEQ ID NO:429), DWQMSSYAGK (SEQ ID NO:430), EMSSYK (SEQ IDNO:431), EMSSYAK (SEQ ID NO:432), EMSSYAGK (SEQ ID NO:433), EQMSSYK (SEQID NO:434), EQMSSYAK (SEQ ID NO:435), EQMSSYAGK (SEQ ID NO:436),EWQMSSYK (SEQ ID NO:437), EWQMSSYAK (SEQ ID NO:438), EWQMSSYAGK (SEQ IDNO:439), MSSYA (SEQ ID NO:440), MSSYAG (SEQ ID NO:441), QMSSY (SEQ IDNO:442), QMSSYA (SEQ ID NO:443), QMSSYAG (SEQ ID NO:444), WQMSSY (SEQ IDNO:445), WQMSSYA (SEQ ID NO:446) and WQMSSYAG (SEQ ID NO:447).

Within other aspects, a modulating agent may comprise an antibody orantigen-binding fragment thereof that specifically binds to theclaudin-7 CAR sequence WQMSSYAG (SEQ ID NO:394).

Within further aspects, a modulating agent comprises one or moreclaudin-8 CAR sequences selected from the group consisting of: VSAF (SEQID NO:5), VSAFI (SEQ ID NO:51), VSAFIE (SEQ ID NO:448), RVSAF (SEQ IDNO:53), RVSAFI (SEQ ID NO:54), RVSAFIE (SEQ ID NO:449), WRVSAF (SEQ IDNO:56), WRVSAFI (SEQ ID NO:57) and WRVSAFIE (SEQ ID NO:450). Such CARsequences may be present within a cyclic peptide, such as: CVSAFC (SEQID NO:217), CVSAFIC (SEQ ID NO:218), CVSAFIEC (SEQ ID NO:451), CRVSAFCCWRVSAFC (SEQ ID NO:223), CWRVSAFIC (SEQ ID NO:224), CWRVSAFIEC (SEQ IDNO:453), KVSAFD (SEQ ID NO:226), KVSAFID (SEQ ID NO:227), KVSAFIED (SEQID NO:454), KRVSAFD (SEQ ID NO:229), KRVSAFID (SEQ ID NO:230), KRVSAFIED(SEQ ID NO:455), KWRVSAFD (SEQ ID NO:232), KWRVSAFID (SEQ ID NO:233),KWRVSAFIED (SEQ ID NO:456), KVSAFE (SEQ ID NO:235), KVSAFIE (SEQ IDNO:236), KVSAFIEE (SEQ ID NO:457), KRVSAFE (SEQ ID NO:238), KRVSAFIE(SEQ ID NO:239), KRVSAFIEE (SEQ ID NO:458), KWRVSAFE (SEQ ID NO:241),KWRVSAFIE (SEQ ID NO:242), KWRVSAFIEE (SEQ ID NO:459), DVSAFK (SEQ IDNO:244), DVSAFIK (SEQ ID NO:245), DVSAFIEK (SEQ ID NO:460), DRVSAFK (SEQID NO:247), DRVSAFIK (SEQ ID NO:248), DRVSAFIEK (SEQ ID NO:461),DWRVSAFK (SEQ ID NO:250), DWRVSAFIK (SEQ ID NO:251), DWRVSAFIEK (SEQ IDNO:462), EVSAFK (SEQ ID NO:253), EVSAFIK (SEQ ID NO:254), EVSAFIEK (SEQID NO:463), ERVSAFK (SEQ ID NO:256), ERVSAFIK (SEQ ID NO:257), ERVSAFIEK(SEQ ID NO:464), EWRVSAFK (SEQ ID NO:259), EWRVSAFIK (SEQ ID NO:260),EWRVSAFIEK (SEQ ID NO:465), VSAFI (SEQ ID NO:262), VSAFIE (SEQ IDNO:466), RVSAF (SEQ ID NO:264), RVSAFI (SEQ ID NO:265), RVSAFIE (SEQ IDNO:467), WRVSAF (SEQ ID NO:267), WRVSAFI (SEQ ID NO:268) and WRVSAFIE(SEQ ID NO:468).

Within other aspects, a modulating agent may comprise an antibody orantigen-binding fragment thereof that specifically binds to theclaudin-8 CAR sequence WRVSAFIE (SEQ ID NO:450).

The present invention further provides pharmaceutical compositionscomprising a cell adhesion modulating agent as described above, incombination with a pharmaceutically acceptable carrier. Suchcompositions may further comprise a drug. In addition, or alternatively,such compositions may further comprise one or more of: (a) a peptidecomprising a cell adhesion recognition sequence that is bound by anadhesion molecule other than a claudin; and/or (b) an antibody orantigen-binding fragment thereof that specifically binds to a celladhesion recognition sequence bound by an adhesion molecule other than aclaudin.

Within further aspects, methods are provided for modulating celladhesion, comprising contacting a claudin-expressing cell with a celladhesion modulating agent as described above.

Within one such aspect, the present invention provides methods forincreasing vasopermeability in a mammal, comprising administering to amammal a cell adhesion modulating agent as provided above, wherein themodulating agent inhibits claudin-mediated cell adhesion.

Within another aspect, methods are provided for reducing unwantedcellular adhesion in a mammal, comprising administering to a mammal acell adhesion modulating agent as provided above wherein the modulatingagent inhibits claudin-mediated cell adhesion.

In yet another aspect, the present invention provides methods forenhancing the delivery of a drug through the skin of a mammal,comprising contacting epithelial cells of a mammal with a cell adhesionmodulating agent as provided above and a drug, wherein the modulatingagent inhibits claudin-mediated cell adhesion, and wherein the step ofcontacting is performed under conditions and for a time sufficient toallow passage of the drug across the epithelial cells.

The present invention further provides methods for enhancing thedelivery of a drug to a tumor in a mammal, comprising administering to amammal a cell adhesion modulating agent as provided above and a drug,wherein the modulating agent inhibits claudin-mediated cell adhesion.

Within further aspects, the present invention provides methods fortreating cancer in a mammal, comprising administering to a mammal a celladhesion modulating agent as provided above, wherein the modulatingagent inhibits claudin-mediated cell adhesion.

The present invention further provides methods for inhibitingangiogenesis in a mammal, comprising administering to a mammal a celladhesion modulating agent as provided above, wherein the modulatingagent inhibits claudin-mediated cell adhesion.

Within further aspects, the present invention provides methods forenhancing drug delivery to the central nervous system of a mammal,comprising administering to a mammal a cell adhesion modulating agent asprovided above, wherein the modulating agent inhibits claudin-mediatedcell adhesion.

The present invention further provides methods for enhancing woundhealing in a mammal, comprising contacting a wound in a mammal with acell adhesion modulating agent as provided above, wherein the modulatingagent enhances claudin-mediated cell adhesion.

Within a related aspect, the present invention provides methods forenhancing adhesion of foreign tissue implanted within a mammal,comprising contacting a site of implantation of foreign tissue in amammal with a cell adhesion modulating agent as provided above, whereinthe modulating agent enhances claudin-mediated cell adhesion.

The present invention further provides methods for inducing apoptosis ina claudin-expressing cell, comprising contacting a claudin-expressingcell with a cell adhesion modulating agent as provided above, whereinthe modulating agent inhibits claudin-mediated cell adhesion.

The present invention further provides methods for identifying an agentcapable of modulating claudin-mediated cell adhesion. One such methodcomprises the steps of (a) culturing cells that express a claudin in thepresence and absence of a candidate agent, under conditions and for atime sufficient to allow cell adhesion; and (b) visually evaluating theextent of cell adhesion among the cells.

Within another embodiment, such methods may comprise the steps of: (a)culturing normal rat kidney cells in the presence and absence of acandidate agent, under conditions and for a time sufficient to allowcell adhesion; and (b) comparing the level of cell surface claudin andE-cadherin for cells cultured in the presence of candidate agent to thelevel for cells cultured in the absence of candidate agent.

Within a further embodiment, such methods may comprise the steps of: (a)culturing human aortic endothelial cells in the presence and absence ofa candidate agent, under conditions and for a time sufficient to allowcell adhesion; and (b) comparing the level of cell surface claudin andN-cadherin for cells cultured in the presence of candidate agent to thelevel for cells cultured in the absence of candidate agent.

Within yet another embodiment, such methods comprise the steps of: (a)contacting an antibody that binds to a modulating agent comprising aclaudin CAR sequence with a test compound; and (b) detecting the levelof antibody that binds to the test compound.

The present invention further provides methods for detecting thepresence of claudin-expressing cells in a samples comprising: (a)contacting a sample to with an antibody that binds to a claudincomprising a claudin CAR sequence under conditions and for a timesufficient to allow formation of an antibody-claudin complex; and (b)detecting the level of antibody-claudin complex, and therefrom detectingthe presence of claudin-expressing cells in the sample.

Within further aspects, the present invention provides kits fordetecting is the presence of claudin-expressing cells in a sample,comprising: (a) an antibody that binds to a modulating agent comprisinga claudin CAR sequence; and (b) a detection reagent.

The present invention further provides, within other aspects, kits forenhancing transdermal drug delivery, comprising: (a) a skin patch; and(b) a cell adhesion modulating agent, wherein the modulating agentcomprises a claudin CAR sequence, and wherein the modulating agentinhibits claudin-mediated cell adhesion.

These and other aspects of the invention will become evident uponreference to the following detailed description and attached drawings.All references disclosed herein are hereby incorporated by reference intheir entirety as if each were individually noted for incorporation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides the amino acid sequences of extracellular domain 1 ofrepresentative mammalian claudins, as indicated. The extracellulardomains were predicted by hydrophobicity analysis using theKyte-Doolittle algorithm (Kyte and Doolittle, J. Molecular Biology157:105-132, 1982), from mouse claudin-1 (SEQ ID NO:6), human claudin-1(SEQ ID NO:529), mouse claudin-2 (SEQ ID NO:7), mouse claudin-3 (SEQ IDNO:469); human claudin-3 (SEQ ID NO: 11), rat claudin-3 (SEQ ID NO:12),mouse claudin-4 (SEQ ID NO:9), human claudin-4 (SEQ ID NO:8), Africangreen Monkey claudin-4 (SEQ ID NO:10), mouse claudin-5 (SEQ ID NO:470),human claudin-5 (SEQ ID NO:471), mouse claudin-6 (SEQ ID NO:472), humanclaudin-6 (SEQ ID NO:530), mouse claudin-7 (SEQ ID NO:473), mouseclaudin-8 (SEQ ID NO:474) and mouse/human claudin-9 (SEQ ID NO:531).Sequences were compared using a Clustal W protein sequence alignment.Amino acids are represented by their IUPAC amino acid codes, where X isany amino acid and—represents a gap. The consensus sequence (SEQ IDNO:13) is shown in italics, and amino acids capitalized within theconsensus represent identity. The claudin family cell adhesionrecognition region is shown in bold.

FIGS. 2A and 2B provide the structures of representative cyclic peptidemodulating agents (SEQ ID NOS: 59-62, 65, 111, 164, 217).

FIG. 3 is a histogram depicting the mean electrical resistance acrossMDCK cell monolayers cultured for 18 hours in medium alone (Control) ormedium containing N—Ac—WKIYSYAGDN—NH₂ (Peptide 118; SEQ ID NO:475) orH—WKIYSYAGDN—NH₂ (Peptide 119; SEQ ID NO:475) at a concentration of 0.5mg/ml. Duplicate measurements were taken, and error bars represent thestandard deviation.

FIG. 4 is a histogram depicting the mean electrical resistance acrossMDCK cell monolayers cultured for 24 hours in medium alone (Control) ormedium containing N—Ac-WKIYSYAGDN-NH₂ (Peptide 118; SEQ ID NO:475) atvarious concentrations.

FIG. 5 provides the amino acid sequences of extracellular domain 1 offurther representative mammalian claudins, as indicated. Theextracellular domains were predicted by hydrophobicity analysis usingthe Kyte-Doolittle algorithm (Kyte and Doolittle, J. Molecular Biology157:105-132, 1982), from mouse claudin-10 (SEQ ID NO:520), humanclaudin-10 (SEQ ID NO:521), mouse claudin-11 (SEQ ID NO:522), humanclaudin-11 (SEQ ID NO:523); mouse claudin-13 (SEQ ID NO:524), mouseclaudin-14 (SEQ ID NO:525), human claudin-14 (SEQ ID NO:526), humanclaudin-15 (SEQ ID NO:527) and bull claudin-16 (SEQ ID NO:528).Sequences were compared using a Clustal W protein sequence alignment.Amino acids are represented by their IUPAC amino acid codes, where X isany amino acid and—represents a gap. The claudin family cell adhesionrecognition region is shown in bold.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention provides cell adhesion modulatingagents comprising peptides that are capable of modulatingclaudin-mediated processes, such as cell adhesion. The present inventionis based on the identification of previously unknown cell adhesionrecognition (CAR) sequences present in claudins. A modulating agent maygenerally comprise one or more claudin CAR sequences), (or analogues ormimetics thereof), with or without one or more additional CAR sequences,as described below. Peptide CAR sequences may be present within a linearor cyclic peptide. Alternatively, or in addition, a modulating agent maycomprise a polynucleotide encoding a peptide comprising one or moreclaudin CAR sequences and/or a modulating agent may comprise a substance(such as an antibody or antigen-binding fragment thereof) thatspecifically binds to a claudin CAR sequence.

In general, to modulate claudin-mediated cell adhesion, aclaudin-expressing cell is contacted with a cell adhesion modulatingagent (also referred to herein as a “modulating agent”) either in vivoor in vitro. Claudin-expressing cells may be readily identified usingany of a variety of techniques well known in the art (such as, forexample, hybridization, PCR or immunohistochemical techniques), andinclude endothelial and epithelial cells, as well as cancer cells, suchas carcinoma cells. Within certain aspects, the methods provided hereininhibit a claudin-mediated function. Such methods include, for example,methods for treating diseases or other conditions characterized byundesirable cell adhesion or for facilitating drug delivery to aspecific tissue or rumor. Certain methods may inhibit cell adhesion(e.g., cancer cell adhesion), as well as cancer invasion and metastasis.Alternatively, a modulating agent may, such as when linked to a matrixor to another modulating agent via a linker, be used to enhance aclaudin-mediated function, such as cell adhesion. Such conjugates may beused, for example, to facilitate wound healing or the adhesion ofimplants.

Cell Adhension Modulating Agents

The term “cell adhesion modulating agent,” as used herein, refers to amolecule comprising at least one of the following components:

(a) a linear or cyclic peptide sequence that is at least 50% identicalto a claudin CAR sequence (i.e., a claudin CAR sequence or an analoguethereof that retains at least 50% identity);

(b) a mimetic (e.g., peptidomimetic or small molecule mimic) of aclaudin CAR sequence;

(c) a substance such as an antibody or antigen-binding fragment thereofthat specifically binds a claudin CAR sequence; and/or

d) a polynucleotide encoding a polypeptide that comprises a claudin CARsequence or analogue thereof.

A modulating agent may consist entirely of one or more of the aboveelements, or may additionally comprise further peptide and/ornon-peptide regions. Additional peptide regions may be derived from aclaudin (preferably an extracellular domain that comprises a CARsequence) and/or may be heterologous. Within certain preferredembodiments, a modulating agent contains no more than 85 consecutiveamino acid residues, and preferably no more than 50 consecutive aminoacid residues, present within a claudin.

A modulating agent is further capable of modulating a function mediatedby a claudin. Such activity may generally be assessed using, forexample, representative assays provided herein. Certain modulatingagents inhibit an interaction between claudin molecules and/or between aclaudin and a different adhesion molecule. For functions (e.g., celladhesion) that are inhibited by a full length, soluble claudin, such amodulating agent may inhibit the function with an activity that is notsubstantially diminished relative to the full length claudin (i.e., themodulating agent inhibits the function at least as well as solubleclaudin, when contacted with cells that express the claudin). Forexample, a modulating agent may be as effective as soluble claudin inpreventing and/or disrupting adhesion of claudin-expressing cells.Alternatively, to enhance adhesion of claudin-expressing cells, amodulating agent may comprise an antibody or antigen-binding fragmentthereof and/or multiple peptides or mimetics linked to a supportmaterial. Such modulating agents may function as a biological glue tobind claudin-expressing cells, and should result in a detectableenhancement of cell adhesion (preferably an enhancement that is at leastas great as that observed for immobilized claudin or antibody directedagainst the claudin).

The term “claudin,” as used herein, refers to an integral membraneprotein with a molecular weight of approximately 22 kD, which containstwo extracellular domains and four transmembrane domains (as determinedby hydrophobicity analysis), and which displays at least 30% sequenceidentity to a member of the claudin family specifically recited herein.Claudins include claudin-1 and claudin-2 (Furuse et al., J. Cell Biology141:1539-1550, 1998; GenBank Accession No. AF115546), which show 38%sequence identity and are present in tight junctions. Other proteinsthat are considered members of the claudin family are claudin-3 (alsoknown as RVP-1; Morita et al., Proc. Natl. Acad Sci. USA 96:511-516,1999; Briehl and Miesfeld, Molecular Endocrinology 5:1381-1388, 1991;Katahira et al., J. Biological Chemistry 272:26652-26656, 1997),claudin-4 (also known as the Clostridium perfringens enterotoxinreceptor (CPE-R); see Morita et al., Proc. Natl. Acad Sci. USA96:511-516, 1999; Katahira et al., J. Cell Biology 136:1239-1247, 1997;Katahira et al., J. Biological Chemistry 272:26652-26656, 1997) andTMVCF (transmembrane protein deleted in Velo-cardio-facial syndrome,also known as claudin-5; Morita et al., Proc. Natl. Acad. Sci. USA96:511-516, 1999; Sirotkin et al. Genomics 42:245-51, 1997), as well asclaudins-6, -7, -8 (Morita et al., Proc. Natl. Acad Sci. USA 96:511-516,1999), all of which have sequences that are 40-60% identical toclaudin-1. Also included is claudin-9 (Tsukita et al., Trends Cell Biol.9: 268-273, 1999; GenBank Accession No. AJ130941). The sequence of thefirst extracellular domain of each of these proteins is shown in FIG. 1.Sequences of the first extracellular domains of other claudins are shownin FIG. 5, including claudin-10 (Tsukita et al., Trends Cell Biol.9:268-273, 1999; GenBank Accession No. NM006984), claudin-11 (Morita etal., J. Cell Biol. 145:579-588, 1999; GenBank Accession No. AJ245901),claudin-13 (Tsukita et al., Trends Cell Biol. 9:268-273, 1999),claudin-14 (Tsukita et al., Trends Cell Biol. 9: 268-273, 1999; GenBankAccession No. AJ132445, claudin-15 (Tsukita et al., Trends Cell Biol.9:268-273, 1999; GenBank Accession No. AJ245738) and claudin-16 (GenBankAccession No. AB030082). All of these proteins, as well as homologuesfrom other species, are considered claudins within the context of thepresent invention, and all contain a claudin CAR sequence, as describedherein.

A claudin CAR sequence, as used herein, is an amino acid sequence thatis present in a naturally occurring claudin and that is capable ofdetectably modulating a claudin-mediated function, such as celladhesion, as described herein. In other words, contacting aclaudin-expressing cell with a peptide comprising a CAR sequence resultsin a detectable change in a claudin-mediated function using at least oneof the representative assays provided herein. CAR sequences may be ofany length, but generally comprise at least three amino acid residues,preferably 4-16 amino acid residues, and more preferably 5-8 amino acidresidues. A peptide modulating agent may comprise any number of aminoacid residues, but preferred agents comprise 3-50 residues, preferably4-16 residues. Within certain embodiments, a peptide modulating agentpreferably comprises an N-acetyl group (i.e., the amino group present onthe amino terminal residue of the peptide is acetylated). It has beenfound, within the context of the present invention, that the presence ofsuch an acetyl group may enhance peptide modulating activity for certainapplications.

Claudin CAR sequences are generally physically located within theclaudin molecule in or near the binding site of an adhesion molecule(i.e., within 10 amino acids, and preferably within 5 amino acids). Thelocation of a binding site may generally be determined using well knowntechniques, such as evaluating the ability of a portion of the claudinto bind to the same claudin or to another adhesion molecule. Anystandard binding assay may be employed for such an evaluation.Recognition of a CAR sequence by the claudin or other adhesion moleculeresults in a measurable effect on an adhesion molecule function, such ascell adhesion. Peptides comprising a CAR sequence generally inhibit sucha function unless linked, as described herein, to form an enhancer ofadhesion molecule function.

It has been found, within the context of the present invention, thatcertain claudin CAR sequences share the consensus sequence:

Trp-Lys/Arg/Gln-Aaa-Baa-Ser/Ala-Tyr/Phe-Caa-Gly/Asp/Glu (SEQ ID NO:1).

Within the consensus sequence, Aaa, Baa and Caa indicate independentlyselected amino acid residues; Lys/Arg/Gln is an amino acid that islysine, arginine or glutamine; Ser/Ala is an amino acid that is serineor alanine; Tyr/Phe is an amino acid that is tyrosine or phenylalanine;and Gly/Asp/Glu is an amino acid that is glycine, aspartic acid orglutamic acid. Representative claudin CAR sequences are provided withinTable I. CAR sequences specifically provided herein further includeportions of such representative CAR sequences, as well as longerpolypeptides that comprise at least a portion of such sequences.Additional claudin CAR sequences may be identified based on sequencehomology to the claudin CAR sequences provided herein, and based on theability of a peptide comprising such a sequence to modulate aclaudin-mediated function within a representative assay describedherein. Within certain embodiments, a modulating agent comprises atleast three consecutive residues, preferably at least five consecutiveresidues and more preferably at least seven consecutive residues, of aclaudin CAR sequence that satisfies the above consensus sequence.

TABLE I Representative Claudin CAR Sequences Claudin CAR Sequence Mouseclaudin-1 WKIYSYAG (SEQ ID NO:34) Human claudin-1 WRIYSYAG (SEQ IDNO:485) Mouse claudin-2 WRTSSYVG (SEQ ID NO:42) Mouse claudin-3 WRVSAFIG(SEQ ID NO:58) Human claudin-3 WRVSAFIG (SEQ ID NO:58) Rat claudin-3WRVSAFIG (SEQ ID NO:58) Human claudin-4 WRVTAFIG (SEQ ID NO:50) Mouseclaudin-4 WRVTAFIG (SEQ ID NO:50) C. aethiops claudin-4 WRVTAFIG (SEQ IDNO:50) Mouse claudin-5 WQVTAFLD (SEQ ID NO: 277) Human claudin-5WQVTAFLD (SEQ ID NO:277) Mouse claudin-6 WKVTAFIG (SEQ ID NO: 335) Humanclaudin-6 WKVTAFIG (SEQ ID NO:335) Mouse claudin-7 WQMSSYAG (SEQ IDNO:394) Mouse claudin-8 WRVSAFIE (SEQ ID NO:450) Mouse claudin-9WKVTAFIG (SEQ ID NO:335) Human claudin-9 WKVTAFIG (SEQ ID NO:335)CONSENSUS Wkxxafxg (SEQ ID NO:1) q sy d r e

Certain preferred claudin CAR sequences comprise 3-8 amino acid residuesof a sequence provided in Table I. For example, a CAR sequence maycomprise 3, 4 or 5 residues of an eight-amino acid sequence in Table I.Certain preferred CAR sequences comprise at least the sequence IYSY (SEQID NO:2), TSSY (SEQ ID NO:3), VTAF (SEQ ID NO:4), MSSY (SEQ ID NO:386)or VSAF (SEQ ID NO:5). A CAR sequence may further comprise one or moreamino acids that flank the sequences provided in Table I, such that theCAR sequence is nine or more amino acids in length. Althoughrepresentative claudin CAR sequences from the claudins presented inTable I are described in more detail below, it will be apparent that CARsequences may be similarly derived from other claudins, such as thoseprovided in FIG. 5.

Representative claudin-1 CAR sequences include IYSY (SEQ ID NO:2), IYSYA(SEQ ID NO:27), KIYSY (SEQ ID NO:29), IYSYAG (SEQ ID NO:28), KIYSYA (SEQID NO:30), WKIYSY (SEQ ID NO:32), KIYSYAG (SEQ ID NO:31), WKIYSYA (SEQID NO:33), WKIYSYAG (SEQ ID NO:34), WKIYSYAGN (SEQ ID NO:476), RIYSY(SEQ ID NO:480), RIYSYA (SEQ ID NO:481), WRIYSY (SEQ ID NO:482), RIYSYAG(SEQ ID NO:483), WRIYSYA (SEQ ID NO:484), WRIYSYAG (SEQ ID NO:485) andWRIYSYAGN (SEQ ID NO:486). Linear peptides having such sequences may bemodified at the N- and/or C-terminal, as in the peptidesN—Ac-WKIYSYAG-NH₂ (SEQ ID NO:34) and N—Ac-WRIYSYAG-NH₂ (SEQ ID NO:485).

Representative claudin-2 CAR sequences include TSSY (SEQ ID NO:3), TSSYV(SEQ ID NO:35), RTSSY (SEQ ID NO:37), TSSYVG (SEQ ID NO:36), RTSSYV (SEQID NO:38), WRTSSY (SEQ ID NO:40), RTSSYVG (SEQ ID NO:39), WRTSSYV (SEQID NO:41), WRTSSYVG (SEQ ID NO:42). Linear peptides having suchsequences may be modified at the N- and/or C-termini, as in the peptideN—Ac-WRTSSYVG-NH₂ (SEQ ID NO:42).

Representative claudin-3 CAR sequences include VSAF (SEQ ID NO:4), VSAFI(SEQ ID NO:51), YSAFIG (SEQ ID NO:52), RVSAF (SEQ ID NO:53), RVSAFI (SEQID NO:54), RVSAFIG (SEQ ID NO:55), WRVSAF (SEQ ID NO:56), WRVSAFI (SEQID NO:57) and WRVSAFIG (SEQ ID NO:58). Linear peptides having suchsequences may be modified at the N- and/or C-termini, as in the peptideN—Ac-WRVSAFIG-NH₂ (SEQ ID NO:58).

Representative claudin-4 CAR sequences include VTAF (SEQ ID NO:4), VTAFI(SEQ ID NO:43), VTAFIG (SEQ ID NO:44), RVTAF (SEQ ID NO:45), RVTAFI (SEQID NO:46), RVTAFIG (SEQ ID NO:47), WRVTAF (SEQ ID NO:48), WRVTAFI (SEQID NO:49) and WRVTAFIG (SEQ ID NO:50). Linear peptides having suchsequences may be modified at the N- and/or C-termini, as in the peptideN—Ac-WRVTAFIG-NH2 (SEQ ID NO:50).

Representative claudin-5 CAR sequences include VTAF (SEQ ID NO:4), VTAFL(SEQ ID NO:270), VTAFLD (SEQ ID NO:271), QVTAF (SEQ ID NO:272), QVTAFL(SEQ ID NO:273), QVTAFLD (SEQ ID NO:274), WQVTAF (SEQ ID NO:275),WQVTAFL (SEQ ID NO:276) and WQVTAFLD (SEQ ID NO:277). Linear peptideshaving such sequences may be modified at the N- and/or C-termini, as inthe peptide N—Ac-WQVTAFLD-NH₂ (SEQ ID NO:277).

Representative claudin-6 and claudin-9 CAR sequences include VTAF (SEQID NO:4), VTAFI (SEQ ID NO:328), VTAFIG (SEQ ID NO:329), KVTAF (SEQ IDNO:330), KVTAFI (SEQ ID NO:331), KVTAFIG (SEQ ID NO:332), WKVTAF (SEQ IDNO:333), WKVTAFI (SEQ ID NO:334) and WKVTAFIG (SEQ ID NO:335). Linearpeptides having such sequences may be modified at the N- and/orC-termini, as in the peptide N—Ac-WKVTAFIG-NH₂ (SEQ ID NO:335).

Representative claudin-7 CAR sequences include MSSY (SEQ ID NO:386),MSSYA (SEQ ID NO:387), MSSYAG (SEQ ID NO:388), QMSSY (SEQ ID NO:389),QMSSYA (SEQ ID NO:390), QMSSYAG (SEQ ID NO:391), WQMSSY (SEQ ID NO:392),WQMSSYA (SEQ ID NO:393) and WQMSSYAG (SEQ ID NO:394). Linear peptideshaving such sequences may be modified; at the N- and/or C-termini, as inthe peptide N—Ac-WQMSSYAG-NH₂ (SEQ ID NO:394).

Representative claudin-8 CAR sequences include VSAF (SEQ ID NO:5), VSAFI(SEQ ID NO:51), VSAFIE (SEQ ID NO:448), RVSAF (SEQ ID NO:53), RVSAFI(SEQ ID NO:54), RVSAFIE (SEQ ID NO:449), WRVSAF (SEQ ID NO:56), WRVSAFI(SEQ ID NO:57) and WRVSAFE (SEQ ID NO:450). Linear peptides having suchsequences may be modified at the N- and/or C-termini, as in the peptideN—Ac-WRVSAFIE-NH₂ (SEQ ID NO:450).

Those of ordinary skill in the art will recognize that similar peptidesequences may be designed to modulate a function mediated by otherclaudins, following identification of a CAR sequence as describedherein.

Certain of the peptide sequences provided above may modulate a functionmediated by multiple claudins. In general, peptides comprising a greaternumber of consecutive residues derived from a particular claudin have agreater specificity for that claudin. In addition, further flankingsequences may be included to enhance specificity. Such flankingsequences may be identified, for example, based on the sequencesprovided in FIG. 1, or based on published sequences. To achievespecificity (i.e., modulation of a particular claudin function that isenhanced relative to the modulation of a function mediated by adifferent claudin), the addition of 2 to 5 flanking residues (preferablyat least one residue on either side of the CAR sequence) is generallysufficient.

As noted above, certain preferred modulating agents comprise a peptide(containing a claudin CAR sequence or an analogue thereof) in which atleast one terminal amino acid residue is modified (e.g., the N-terminalamino group is modified by, for example, acetylation oralkoxybenzylation and/or an amide or ester is formed at the C-terminus).It has been found, within the context of the present invention, that theaddition of at least one such group to a linear or cyclic peptidemodulating agent may improve the ability of the agent to modulate aclaudin-mediated function. Certain preferred modulating agents containmodifications at the N- and C-terminal residues, such asN—Ac-WKIYSYAG-NH₂ (SEQ ID NO:34) or N—Ac-WKIYSYAGN-NH₂ (SEQ ID NO:476),which modulates claudin-1 mediated functions. Other CAR sequencesprovided herein are also preferably modified by the addition of one ormore terminal groups.

The present invention further contemplates claudin CAR sequences fromother organisms. Such CAR sequences may be identified based uponsequence similarity to the sequences provided herein, and the ability tomodulate a claudin-mediated function such as may be confirmed asdescribed herein.

As noted above, modulating agents as described herein may comprise ananalogue or mimetic of a claudin CAR sequence. An analogue generallyretains at least 50% identity to a native claudin CAR sequence, andmodulates a claudin-mediated function as described herein. Suchanalogues preferably contain at least three residues of, and morepreferably at least five residues of, a claudin CAR sequence. Ananalogue may contain any of a variety of amino acid substitutions,additions, deletions and/or modifications (e.g., side chainmodifications). Preferred amino acid substitutions are conservative. A“conservative substitution” is one in which an amino acid is substitutedfor another amino acid that has similar properties, such that oneskilled in the art of peptide chemistry would expect the secondarystructure and hydropathic nature of the polypeptide to be substantiallyunchanged. Amino acid substitutions may generally be made on the basisof similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity and/or the amphipathic nature of the residues. Forexample, negatively charged amino acids include aspartic acid andglutamic acid; positively charged amino acids include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine and valine;glycine and alanine, asparagine and glutamine; and serine, threonine,phenylalanine and tyrosine. Other groups of amino acids that mayrepresent conservative changes include: (1) ala, pro, gly, glu, asp,gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala,phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. The criticaldetermining: feature of a claudin CAR sequence analogue is the abilityto modulate a claudin-mediated function, which may be evaluated usingthe representative assays provided herein.

A mimetic is a non-peptidyl compound that is conformationally similar toa claudin CAR sequence, such that it modulates a claudin-mediatedfunction as described below. Such mimetics may be designed based ontechniques that evaluate the three dimensional structure of the peptide.For example, Nuclear Magnetic Resonance spectroscopy (NMR) andcomputational techniques may be used to determine the conformation of aclaudin CAR-sequence. NMR is widely used for structural analyses of bothpeptidyl and non-peptidyl compounds. Nuclear Overhauser Enhancements(NOE's), coupling constants and chemical shifts depend on theconformation of a compound. NOE data provides the interproton distancebetween protons through space and can be used to calculate the lowestenergy conformation for the claudin CAR sequence. This information canthen be used to design mimetics of the preferred conformation. Linearpeptides in solution exist in many conformations. By usingconformational restriction techniques it is possible to fix the peptidein the active conformation. Conformational restriction can be achievedby i) introduction of an alkyl group such as a methyl which stericallyrestricts free bond rotation; ii) introduction of unsaturation whichfixes the relative positions of the terminal and geminal substituents;and/or iii) cyclization, which fixes the relative positions of thesidechains. Mimetics may be synthesized where one or more of the amidelinkages has been replaced by isosteres, substituents or groups whichhave the same size or volume such as —CH₂NH—, —CSNH—, —CH₂S—, —CH═CH—,—CH₂CH₂—, —CONMe— and others. These backbone amide linkages can also bepart of a ring structure (e.g., lactam). Mimetics may be designed whereone or more of the side chain functionalities of the claudin CARsequence are replaced by groups that do not necessarily have the samesize or volume, but have similar chemical and/or physical propertieswhich produce similar biological responses. Other mimetics may be smallmolecule mimics, which may be readily identified from small moleculelibraries, based on the three-dimensional structure of the CAR sequence.It should be understood that, within embodiments described below, ananalogue or mimetic may be substituted for a claudin CAR sequence.

Modulating agents, or peptide portions thereof, may be linear or cyclicpeptides. The term “cyclic peptide,” as used herein, refers to a peptideor salt thereof that comprises (1) an intramolecular covalent bondbetween two non-adjacent residues, forming a peptide ring and (2) atleast one claudin CAR sequence or an analogue thereof present within thepeptide ring. The intramolecular bond may be a backbone to backbone,side-chain to backbone or side-chain to side-chain bond (i.e., terminalfunctional groups of a linear peptide and/or side chain functionalgroups of a terminal or interior residue may be linked to achievecyclization). Preferred intramolecular bonds include, but are notlimited to, disulfide, amide and thioether bonds. Any of the aboveclaudin CAR sequences, or an analogue or mimetic thereof, may beincorporated into a cyclic peptide, with or without one or more otheradhesion molecule CAR sequences. Additional adhesion molecule CARsequences are described in greater detail below.

The size of a cyclic peptide ring generally ranges from 5 to about 15residues, preferably from 5 to 10 residues. Additional residue(s) may bepresent on the N-terminal and/or C-terminal side of a claudin CARsequence, and may be derived from sequences that flank a CAR sequence,with or without amino acid substitutions and/or other modifications.Alternatively, additional residues present on one or both sides of theCAR sequence(s) may be unrelated to an endogenous sequence (e.g.,residues that facilitate cyclization, purification or other manipulationand/or residues having a targeting or other function).

Within certain embodiments, a modulating agent may comprise a cyclicpeptide that contains a claudin CAR sequence as provided in Table I (ora portion of such a CAR sequence). Certain cyclic peptides have theformula:

Within this formula, W is a tetrapeptide selected from the groupconsisting of IYSY (SEQ ID NO:2), TSSY (SEQ ID NO:3), VTAF (SEQ IDNO:4), MSSY (SEQ ID NO:386) and VSAF (SEQ ID NO:5); X₁, and X₂ areoptional, and if present, are independently selected from the groupconsisting of amino acid residues and combinations thereof in which theresidues are linked by peptide bonds, and wherein X₁ and X₂independently range in size from 0 to 10 residues, such that the sum ofresidues contained within X₁ and X₂ ranges from 1 to 12; Y₁ and Y₂ areindependently selected from the group consisting of amino acid residues,and wherein a covalent bond is formed between residues Y₁ and Y₂; and Z₁and Z₂ are optional, and if present, are independently selected from thegroup consisting of amino acid residues and combinations thereof inwhich the residues are linked by peptide bonds.

Cyclic peptides may comprise any of the above CAR sequence(s). Suchcyclic peptides may be used as modulating agents without modification,or may be incorporated into a modulating agent. For example, a cyclicpeptide may comprise any of the above claudin-1 CAR sequences.Representative cyclic peptides include CIYSYC (SEQ ID NO:59), CIYSYAC(SEQ ID NO:60), CIYSYAGC (SEQ ID NO:61), CKIYSYC (SEQ ID NO:62),CKIYSYAC (SEQ ID NO:63), CKIYSYAGC (SEQ ID NO:64), CWKIYSYC (SEQ IDNO:65), CWKIYSYAC (SEQ ID .NO:66), CWKIYSYAGC (SEQ ID NO:67), CRIYSYC(SEQ ID NO:487), CRIYSYAC (SEQ ID NO:488), CRIYSYAGC (SEQ ID NO:489),CWRIYSYC (SEQ ID NO:490), CWRIYSYAC (SEQ ID NO:491), CWRIYSYAGC (SEQ IDNO:492), KIYSYD (SEQ ID NO:68), KIYSYAD (SEQ ID NO:69), KIYSYAGD (SEQ IDNO:70), KKIYSYD (SEQ ID NO:71), KKIYSYAD (SEQ ID NO:72), KKIYSYAGD (SEQID NO:73), KWKIYSYD (SEQ ID NO:74), KWKIYSYAD (SEQ ID NO:75), KWKIYSYAGD(SEQ ID NO:76), KRIYSYD (SEQ ID NO:493), KRIYSYAD (SEQ ID NO:494),KRIYSYAGD (SEQ ID NO:495), KWRIYSYD (SEQ ID NO:496), KWRIYSYAD (SEQ IDNO:497), KWRIYSYAGD (SEQ ID NO:498), KIYSYE (SEQ ID NO:77), KIYSYAE (SEQID NO:78), KIYSYAGE (SEQ ID NO:79), KKIYSYE (SEQ ID NO:80), KKIYSYAE(SEQ ID NO:81), KKIYSYAGE (SEQ ID NO:82), KWKIYSYE (SEQ ID NO:83),KWKlYSYAE (SEQ ID NO:84), KWKIYSYAGE (SEQ ID NO:85), KRIYSYE (SEQ IDNO:499), KRIYSYAE (SEQ ID NO:500), KRIYSYAGE (SEQ ID NO:501), KWRIYSYE(SEQ ID NO:502), KWRIYSYAE (SEQ ID NO:503), KWRIYSYAGE (SEQ ID NO:504),DIYSYK (SEQ ID NO:86), DIYSYAK (SEQ ID NO:87), DIYSYAGK (SEQ ID NO:88),DKIYSYK (SEQ ID NO:89), DKIYSYAK (SEQ ID NO:90), DKIYSYAGK (SEQ IDNO:91), DWKISYK (SEQ ID NO:92), DWKISYAK (SEQ ID NO:93), DWKISYAGK (SEQID NO:94),DRIYSYK (SEQ ID NO:505), DRIYSYAK (SEQ ID NO:506), DRIYSYAGK(SEQ ID NO:507), DWRIYSYK (SEQ ID NO:508), DWRIYSYAK (SEQ ID NO:509),DWRIYSYAGK (SEQ ID NO:510), EIYSYK (SEQ ID NO:95), EIYSYAK (SEQ IDNO:96), EIYSYAGK (SEQ ID NO:97), EKIYSYK (SEQ ID NO:98), EKIYSYAK (SEQID NO:99), EKIYSYAGK (SEQ ID NO:100), EWKIYSYK (SEQ ID NO:101),EWKIYSYAK (SEQ ID NO:102), EWKIYSYAGK (SEQ ID NO:103), ERIYSYK (SEQ IDNO:511), ERIYSYAK (SEQ ID NO:512), ERIYSYAGK (SEQ ID NO:513), EWRIYSYK(SEQ ID NO:514), EWRIYSYAK (SEQ ID NO:515), EWRIYSYAGK (SEQ ID NO:516),IYSYA (SEQ ID NO:104), IYSYAG (SEQ ID NO:105), KIYSY (SEQ ID NO:106),KIYSYAG (SEQ ID NO:107), WKIYSY (SEQ ID NO:108), WKIYSYA (SEQ IDNO:109), WKIYSYAG (SEQ ID NO:110), RIYSY (SEQ ID NO:517), WRIYSYA (SEQID NO:518) and WRIYSYAG (SEQ ID NO:519). Within the context of thepresent invention, underlined sequences are cyclized using any suitablemethod, as described herein.

Similarly, cyclic peptides may comprise any of the above claudin-2 CARsequences. Representative cyclic peptides include: CTSSYC (SEQ IDNO:111), CTSSYVC (SEQ ID NO:112), CTSSYVGC (SEQ ID NO:113), CRTSSYC (SEQID NO:114), CRTSSYVC (SEQ ID NO:115), CRTSSYVGC (SEQ ID NO:116),CWRTSSYC (SEQ ID NO:117), CWRTSSYVC (SEQ ID NO: 118), CWRTSSYVGC (SEQ IDNO:119), KTSSYD (SEQ ID NO:120), KTSSYVD (SEQ ID NO:121), KTSSYVGD (SEQID NO:122), KRTSSYD (SEQ ID NO:123), KRTSSYVD (SEQ ID NO:124), KRTSSYVGD(SEQ ID NO:125), KWRTSSYD (SEQ ID NO:126), KWRTSSYVD (SEQ ID NO:127),KWRTSSYVGD (SEQ ID NO:128), KTSSYE (SEQ ID NO:129), KTSSYVE (SEQ IDNO:130), KTSSYVGE (SEQ ID NO:131), KRTSSYE (SEQ ID NO:132), KRTSSYVE(SEQ ID NO:133), KRTSSYVGE (SEQ ID NO:134), KWRTSSYE (SEQ ID NO:135),KWRTSSYVE (SEQ ID NO:136), KWRTSSYVGE (SEQ ID NO:137), DTSSYK (SEQ IDNO:138), DTSSYVK (SEQ ID NO:139), DTSSYVGK (SEQ ID NO:140), DRTSSYK (SEQID NO:141), DRTSSYVK (SEQ ID NO: 142), DRTSSYVGK (SEQ ID NO: 143),DWRTSSYK (SEQ ID NO:144), DWRTSSYVK (SEQ ID NO:145), DWRTSSYVGK (SEQ IDNO:146), ETSSYK (SEQ ID NO:147), ETSSYVK (SEQ ID NO:148), ETSSYVGK (SEQID NO:149), ERTSSYK (SEQ ID NO:150), ERTSSYVK (SEQ ID NO:151), ERTSSYVGK(SEQ ID NO:152), EWRTSSYK (SEQ ID NO:153), EWRTSSYVK (SEQ ID NO:154),EWRTSSYVGK (SEQ ID NO:155), TSSYV (SEQ ID NO:156), TSSYVG (SEQ IDNO:157), RTSSY (SEQ ID NO:158), RTSSYV (SEQ ID NO:159), RTSSYVG (SEQ IDNO:160), WRTSSY (SEQ ID NO:161), WRTSSYV (SEQ ID NO:162) and WRTSSYVG(SEQ ID NO:163).

Representative cyclic peptides comprising a claudin-3 CAR sequenceinclude: CVSAFC (SEQ ID NO:217), CVSAFIC (SEQ ID NO:218), CVSAFIGC (SEQID NO:219), CRVSAFC (SEQ ID NO:220), CRVSAFIC (SEQ ID NO:221), CRVSAFIGC(SEQ ID NO:222), CWRVSAFC (SEQ ID NO:223), CWRVSAFIC (SEQ ID NO:224),CWRVSAFIGC (SEQ ID NO:225), KVSAFD (SEQ ID NO:226), KVSAFID (SEQ IDNO:227), KVSAFIGD (SEQ ID NO:228), KRVSAFD (SEQ ID NO:229), KRVSAFID(SEQ ID NO:230), KRVSAFIGD (SEQ ID NO:231), KWRVSAFD (SEQ ID NO:232),KWRVSAFID (SEQ ID NO:233), KWRVSAFIGD (SEQ ID NO:234), KVSAFE (SEQ IDNO:235), KVSAFIE (SEQ ID NO:236), KVSAFIGE (SEQ ID NO:237), KRVSAFE (SEQID NO:238), KRVSAFIE (SEQ ID NO:239), KRVSAFIGE (SEQ ID NO:240),KWRVSAFE (SEQ ID NO:241), KWRVSAFIE (SEQ ID NO:242), KWRVSAFIGE (SEQ IDNO:243), DVSAFK (SEQ ID NO:244), DVSAFIK (SEQ ID NO:245), DVSAFIGK (SEQID NO:246), DRVSAFK (SEQ ID NO:247), DRVSAFIK (SEQ ID NO:248), DRVSAFIGK(SEQ ID NO:249), DWRVSAFK (SEQ ID NO:250), DWRVSAFIK (SEQ ID NO:251),DWRVSAFIGK (SEQ ID NO:252), EVSAFK (SEQ ID NO:253), EVSAFIK (SEQ IDNO:254), EVSAFIGK (SEQ ID NO:255), ERVSAFK (SEQ ID NO:256), ERVSAFIK(SEQ ID NO:257), ERVSAFIGK (SEQ ID NO:258), EWRVSAFK (SEQ ID NO:259),EWRVSAFIK (SEQ ID NO:260), EWRVSAFIGK (SEQ ID NO:261), VSAFI (SEQ IDNO:262), VSAFIG (SEQ ID NO:263), RVSAF (SEQ ID NO:264), RVSAFI (SEQ IDNO:265), RVSAFIG (SEQ ID NO:266), WRVSAF (SEQ ID NO:267), WRVSAFI (SEQID NO:268) and WRVSAFIG (SEQ ID NO:269).

Any claudin-4 CAR sequence(s) may be formulated into a cyclic peptide.Representative cyclic peptides include: CVTAFC (SEQ ID NO:164), CVTAFIC(SEQ ID NO:165), CVTAFIGC (SEQ ID NO:166), CRVTAFC (SEQ ID NO:167),CRVTAFIC (SEQ ID NO:168), CRVTAFIGC (SEQ ID NO:169), CWRVTAFC (SEQ IDNO:170), CWRVTAFIC (SEQ ID NO:171), CWRVTAFIGC (SEQ ID NO:172), KVTAFD(SEQ ID NO:173), KVTAFID (SEQ ID NO:174), KVTAFIGD (SEQ ID NO:175),KRVTAFD (SEQ ID NO:176), KRVTAFID (SEQ ID NO:177), KRVTAFIGD (SEQ IDNO:178), KWRVTAFD (SEQ ID NO:179), KWRVTAFID (SEQ ID NO:180), KWRVTAFIGD(SEQ ID NO:181), KVTAFE (SEQ ID NO:182), KVTAFIE (SEQ ID NO:183),KVTAFIGE (SEQ ID NO:184), KRVTAFE (SEQ ID NO:185), KRVTAFE (SEQ IDNO:186), KRVTAFIGE (SEQ ID NO:187), KWRVTAFE (SEQ ID NO:188), KWRVTAFIE(SEQ ID NO:189), KWRVTAFIGE (SEQ ID NO:190), DVATFK (SEQ ID NO: 191),DVTAFIK (SEQ ID NO:192), DVTAFIGK (SEQ ID NO:193), DRVTAFK (SEQ IDNO:194), DRVTAFIK (SEQ ID NO:195), DRVTAFIGK (SEQ ID NO:196), DWRVTAFK(SEQ ID NO:197), DWRVTAFIK (SEQ ID NO:198), DWRVTAFIGK (SEQ ID NO:199),EVTAFK (SEQ ID NO:200), EVTAFIK (SEQ ID NO:201), EVTAFIGK (SEQ IDNO:202), ERVTAFK (SEQ ID NO:203), ERVTAFIK (SEQ ID NO:204), ERVTAFIGK(SEQ ID NO:205), EWRVTAFK (SEQ ID NO:206), EWRVTAFIK (SEQ ID NO:207),EWRVTAFIGK (SEQ ID NO:208), VTAFI (SEQ ID NO:209), VTAFIG (SEQ IDNO:210), RVTAF (SEQ ID NO:211), RVTAFI (SEQ ID NO:212), RVTAFIG (SEQ IDNO:213), WRVTAF (SEQ ID NO:214), WRVTAFI (SEQ ID NO:215) and WRVTAFIG(SEQ ID NO:216).

Representative cyclic peptides comprising a claudin-5 CAR sequenceinclude: CVTAFC (SEQ ID NO:164), CVTAFLC (SEQ ID NO:278), CVTAFLDC (SEQID NO:279), CQVTAFC (SEQ ID NO:280), CQVTAFLC (SEQ ID NO:281), CQVTAFLDC(SEQ ID NO:282), CWQVTAFC (SEQ ID NO:283), CWQVTAFLC (SEQ ID NO:284),CWQVTAFLDC (SEQ ID NO:285), KVTAFD (SEQ ID NO:286), KVTAFLD (SEQ IDNO:287), KVTAFLDD (SEQ ID NO:288), KQVTAFD (SEQ ID NO:289), KQVTAFLD(SEQ ID NO:290), KQVTAFLDD (SEQ ID NO:291), KWQVTAFD (SEQ ID NO:292),KWQVTAFLD (SEQ ID NO:293), KWQVTAFLDD (SEQ ID NO:294), KVTAFE (SEQ IDNO:182), KVTAFLE (SEQ ID NO:295), KVTAFLDE (SEQ ID NO:296), KQVTAFE (SEQID NO:297), KQVTAFLE (SEQ ID NO:298), KQVTAFLDE (SEQ ID NO:299),KWQVTAFE (SEQ ID NO:300), KWQVTAFLE (SEQ ID NO:301), KWQVTAFLDE (SEQ IDNO:302), DVATFK (SEQ ID NO:303), DVTAFLK (SEQ ID NO:304), DVTAFLDK (SEQID NO:305), DQVTAFK (SEQ ID NO:306), DQVTAFLK (SEQ ID NO:307), DQVTAFLDK(SEQ ID NO:308), DWQVTAFK (SEQ ID NO:309), DWQVTAFLK (SEQ ID NO:310),DWQVTAFLDK (SEQ ID NO:311), EVTAFK (SEQ ID NO:200), EVTAFLK (SEQ IDNO:312), EVTAFLDK (SEQ ID NO:313), EQVTAFK (SEQ ID NO:314), EQVTAFLK(SEQ ID NO:315), EQVTAFLDK (SEQ ID NO:316), EWQVTAFK (SEQ ID NO:317),EWQVTAFLK (SEQ ID NO:318), EWQVTAFLDK (SEQ ID NO:319), VTAFL (SEQ IDNO:320), VTAFLD (SEQ ID NO:321), QVTAF (SEQ ID NO:322), QVTAFL (SEQ IDNO:323), QVTAFLD (SEQ ID NO:324), WQVTAF (SEQ ID NO:325), WQVTAFL (SEQID NO:326) and WQVTAFLD (SEQ ID NO:327).

Representative cyclic peptides comprising a claudin-6 or claudin-9 CARsequence include: CVTAFC (SEQ ID NO:164), CVTAFIC (SEQ ID NO:336),CVTAFIGC (SEQ ID NO:337), CKVTAFC (SEQ ID NO:338), CKVTAFIC (SEQ IDNO:339), CKVTAFIGC (SEQ ID NO:340), CWKVTAFC (SEQ ID NO:341), CWKVTAFIC(SEQ ID NO:342), CWKVTAFIGC (SEQ ID NO:343), KVTAFD (SEQ ID NO:344),KVTAFID (SEQ ID NO:345), KVTAFIGD (SEQ ID NO:346), KKVTAFD (SEQ IDNO:347), KKVTAFID (SEQ ID NO:348), KKVTAFIGD (SEQ ID NO:349), KWKVTAFD(SEQ ID NO:350), KWKVTAFID (SEQ ID NO:351), KWKVTAIGD (SEQ ID NO:352),KVTAFE (SEQ ID NO:182), KVTAFIE (SEQ ID NO:353), KVTAFIGE (SEQ IDNO:354), KKVTAFE (SEQ ID NO:355), KKVTAFIE (SEQ ID NO:356), KKVTAFIGE(SEQ ID NO:357), KWKVTAFE (SEQ ID NO:358), KWKVTAFIE (SEQ ID NO:359),KWKVTAFIGE (SEQ ID NO:360), DVATFK (SEQ ID NO:361), DVTAFIK (SEQ IDNO:362), DVTAFIGK (SEQ ID NO:363), DKVTAFK (SEQ ID NO:364), DKVTAFIK(SEQ ID NO:365), DKVTAFIGK (SEQ ID NO:366), DWKVTAFK (SEQ ID NO:367),DWKVTAFIK (SEQ ID NO:368), DWKVTAFIGK (SEQ ID NO:369), EVTAFK (SEQ IDNO:200), EVTAFIK (SEQ ID NO:370), EVTAFIGK (SEQ ID NO:371), EKVTAFK (SEQID NO:372), EKVTAFIK (SEQ ID NO:373), EKVTAFIGK (SEQ ID NO:374),EWKVTAFK (SEQ ID NO:375), EWKVTAFIK (SEQ ID NO:376), EWKVTAFIGK (SEQ IDNO:377), VTAFI (SEQ ID NO:378), VTAFIG (SEQ ID NO:379), KVTAF (SEQ IDNO:380), KVTAFI (SEQ ID NO:38 1), KVTAFIG (SEQ ID NO:382), WKVTAF (SEQID NO:383), WKVTAFI (SEQ ID NO:384) and WKVTAFIG (SEQ ID NO:385).

Representative cyclic peptides comprising a claudin-7 CAR sequenceinclude: CMSSYC (SEQ ID NO:395), CMSSYAC (SEQ ID NO:396), CMSSYAGC (SEQID NO:397), CQMSSYC (SEQ ID NO:398), CQMSSYAC (SEQ ID NO:399), CQMSSYAGC(SEQ ID NO:400), CWQMSSYC (SEQ ID NO:401), CWQMSSYAC (SEQ ID NO:402),CWQMSSYAGC (SEQ ID NO:403), KMSSYD (SEQ ID NO:404), KMSSYAD (SEQ IDNO:405), KMSSYAGD (SEQ ID NO:406), KQMSSYD (SEQ ID NO:407), KQMSSYAD(SEQ ID NO:408), KQMSSYAGD (SEQ ID NO:409), KWQMSSYD (SEQ ID NO:410),KWQMSSYAD (SEQ ID NO:411), KWQMSSYAGD (SEQ ID NO:412), KMSSYE (SEQ IDNO:413), KMSSYAE (SEQ ID NO:414), KMSSYAGE (SEQ ID NO:415), KQMSSYE (SEQID NO:416), KQMSSYAE (SEQ ID NO:417), KQMSSYAGE (SEQ ID NO:418),KWQMSSYE (SEQ ID NO:419), KWQMSSYAE (SEQ ID NO:420), KWQMSSYAGE (SEQ IDNO:421), DMSSYK (SEQ ID NO:422), DMSSYAK (SEQ ID NO:423), DMSSYAGK (SEQID NO:424), DQMSSYK (SEQ ID NO:425), DQMSSYAK (SEQ ID NO:426), DQMSSYAGK(SEQ ID NO:427), DWQMSSYK (SEQ ID NO:428), DWQMSSYAK (SEQ ID NO:429),DWQMSSYAGK (SEQ ID NO:430), EMSSYK (SEQ ID NO:43 1), EMSSYAK (SEQ IDNO:432), EMSSYAGK (SEQ ID NO:433), EQMSSYK (SEQ ID NO:434), EOMSSYAK(SEQ ID NO:435), EQMSSYAGK (SEQ ID NO:436), EWQMSSYK (SEQ ID NO:437),EWQMSSYAK (SEQ ID NO:438), EWQMSSYAGK (SEQ ID NO:439), MSSYA (SEQ IDNO:440), MSSYAG (SEQ ID NO:441), QMSSY (SEQ ID NO:442), QMSSYA (SEQ IDNO:443), QMSSYAG (SEQ ID NO:444), WQMSSY (SEQ ID NO:445), WQMSSYA (SEQID NO:446) and WQMSSYAG (SEQ ID NO:447).

Representative cyclic peptides comprising a claudin-8 CAR sequenceinclude: CVSAFC (SEQ ID NO:217), CVSAFIC (SEQ ID NO:218), CVSAFIEC (SEQID NO:451), CRVSAFC (SEQ ID NO:220), CRVSAFIC (SEQ ID NO:221), CRVSAFIEC(SEQ ID NO:452), CWRVSAFC (SEQ ID NO:223), CWRVSAFIC (SEQ ID NO:224),CWRVSAFIEC (SEQ ID NO:453), KVSAFD (SEQ ID NO:226), KVSAFID (SEQ IDNO:227), KVSAFIED (SEQ ID NO:454), KRVSAFD (SEQ ID NO:229), KRVSAFID(SEQ ID NO:230), KRVSAFIED (SEQ ID NO:455), KWRVSAFD (SEQ ID NO:232),KWRVSAFID (SEQ ID NO:23:3), KWRVSAIED (SEQ ID NO:456), KVSAFE (SEQ IDNO:235), KYSAFIE (SEQ ID NO:236), KVSAFIEE (SEQ ID NO:457), KRVSAFE (SEQID NO:238), KRVSAFIE (SEQ ID NO:239), KRVSAFIEE (SEQ ID NO:458),KWRVSAFE (SEQ ID NO:241), KWRVSAFFE (SEQ ID NO:242), KWRVSAFIEE (SEQ IDNO:459). DVSAFK (SEQ ID NO:244), DVSAFRK (SEQ ID NO:245), DVSAFIEK (SEQID NO:460), DRVSAFK (SEQ ID NO:247), DRVSAFTK (SEQ ID NO:248), DRVSAFEEK(SEQ ID NO:461), DWRVSAFK (SEQ ID NO:250), DWRVSAFIK (SEQ ID NO:251),DWRVSAFIEK (SEQ ID NO:462), EVSAFK (SEQ ID NO:253), EVSAFIK (SEQ IDNO:254), EVSAFIEK (SEQ ID NO:463), ERVSAFK (SEQ ID NO:256), ERVSAFIK(SEQ ID NO:257), ERVSAFIEK (SEQ ID NO:464), EWRVSAFK (SEQ ID NO:259),EWRVSAFIK (SEQ ID NO:260), EWRVSAFIEK (SEQ ID NO:465), VSAFI (SEQ IDNO:262), VSAFIE (SEQ ID NO:466), RVSAF (SEQ ID NO:264), RVSAFI (SEQ IDNO:265), RVSAFIE (SEQ ID NO:467), WRVSAF (SEQ ID NO:267), WRVSAFI (SEQID NO:268) and WRVSAFIE (SEQ ID NO:468).

Within certain embodiments, as discussed below, cyclic peptides thatcontain small CAR sequences (e.g., four residues without significantflanking sequences) are preferred for modulating claudin-mediatedfunctions. Such peptides may contain an N-acetyl group and a C-amidegroup (e.g., the 6-residue ring N—Ac-CIYSYC-NH₂ (SEQ ID NO:59) orN—Ac-KIYSYD-NH₂ (SEQ ID NO:68), for modulating claudin-1 mediatedfunctions). Small cyclic peptides may generally be used to specificallymodulate adhesion of cancer and/or other cell types by topicaladministration or by systemic administration, with or without linking atargeting agent to the peptide, as discussed below. Certainrepresentative cyclic peptides comprising a claudin CAR sequence areshown in FIGS. 2A and 2B. Within other preferred embodiments, a cyclicpeptide may contain sequences that flank the claudin CAR sequence on oneor both sides, which may result in increased potency. Suitable flankingsequences include, but are not limited to, an endogenous sequencepresent in a naturally occurring claudin. To facilitate the preparationof cyclic peptides having increased potency, nuclear magnetic resonance(NMR) and computational techniques may be used to determine theconformation of a peptide that confers increased potency, as describedabove.

Within embodiments in which inhibition of a claudin interaction isdesired, a modulating agent may contain one claudin CAR sequence, ormultiple CAR sequences that are adjacent to one another (i.e., withoutintervening sequences) or in close proximity (i.e., separated by peptideand/or non-peptide linkers to give a distance between the claudin CARsequences that ranges from about 0.1 to 400 nm). For example, amodulating agent with adjacent IYSY (SEQ ID NO:2) sequences may comprisethe peptide IYSYIYSY (SEQ ID NO: 14). A representative modulating agentwith IYSY (SEQ ID NO:2) sequences in close proximity may comprise thesequence KIYSYKIYSYKIYSY (SEQ ID NO:15). A linker may be any molecule(including peptide and/or non-peptide sequences) that does not contain aCAR sequence and that can be covalently linked to at least two peptidesequences. Using a linker, CAR sequence-containing peptides and otherpeptide or protein sequences may be joined end-to-end (i.e., the linkermay be covalently attached to the carboxyl or amino group of eachpeptide sequence), and/or via side chains. One linker that can be usedfor such purposes is (H₂N(CH₂)_(n)CO₂H), or derivatives thereof, where nranges from 1 to 4. Other linkers that may be used will be apparent tothose of ordinary skill in the art. Peptide and non-peptide linkers maygenerally be incorporated into a modulating agent using any appropriatemethod known in the art.

Within embodiments in which enhancement of cell adhesion mediated by aclaudin is desired, a modulating agent may contain multiple claudin CARsequences, or antibodies that specifically bind to such sequences,joined by linkers as described above. For enhancers of claudin function,the linker distance should generally be 400-10,000 nm. One linker thatcan be used for such purposes is (H₂N(CH₂)_(n)CO₂H)_(m), or derivativesthereof, where n ranges from 1 to 10 and m ranges from 1 to 4000. Forexample, if glycine (H₂NCH₂CO₂H) or a multimer thereof is used as alinker, each glycine unit corresponds to a linking distance of 2.45angstroms, or 0.245 nm, as determined by calculation of its lowestenergy conformation when linked to other amino acids using molecularmodeling techniques. Similarly, aminopropanoic acid corresponds to alinking distance of 3.73 angstroms, aminobutanoic acid to 4.96angstroms, aminopentanoic acid to 6.30 angstroms and amino hexanoic acidto 6.12 angstroms. Enhancement of cell adhesion may also be achieved byattachment of multiple modulating agents to a support material, asdiscussed further below.

A modulating agent as described herein may additionally comprise a CARsequence for one or more different adhesion molecules (including, butnot limited to, other CAMs) and/or one or more antibodies or fragmentsthereof that bind to such sequences. Linkers may, but need not, be usedto separate such CAR sequence(s) and/or antibody sequence(s) from theclaudin CAR sequence(s) and/or each other. Such modulating agents maygenerally be used within methods in which it is desirable tosimultaneously disrupt cell adhesion mediated by multiple adhesionmolecules. For cyclic peptides, additional CAR sequences may be presentwithin the cyclic peptide ring, within a separate cyclic peptidecomponent of the modulating agent and/or in a non-cyclic portion of themodulating agent. Antibodies and antigen-binding fragments thereof aretypically present in a non-cyclic portion of the modulating agent.

As used herein, an “adhesion molecule” is any molecule that mediatescell adhesion via a receptor on the cell's surface. Adhesion moleculesinclude cell adhesion proteins (e.g., classical cadherins, other membersof the cadherin gene superfamily that are not classical cadherins (suchas atypical cadherins (e.g., VE-cadherin and PB-cadherin), desmogleins(Dsg) and desmocollins (Dsc)); integrins; occludin; and members of theimmunoglobulin supergene family, such as N-CAM, JAM and PECAM).Preferred CAR sequences for inclusion within a modulating agent includeHis-Ala-Val (HAV), which is bound by classical cadherins (Munro S B etal., 1996, In: Cell Adhesion and Invasion in Cancer Metastasis, P.Brodt, ed., pp. 17-34 (RG Landes Company, Austin Tex.); Arg-Gly-Asp(RGD), which is bound by integrins (see Cardarelli et al., J. Biol.Chem. 267:23159-64, 1992); KYSFNYDGSE (SEQ ID NO: 16), which is bound byN-CAM; SFTFDPKSG (SEQ ID NO:477) or DPK, which is bound by JAM, theoccludin CAR sequence LYHY (SEQ ID NO:17); and/or one or morenonclassical cadherin CAR sequences, such as the VE-cadherin CARsequence DAE, the Dsc CAR sequences IEK, VER and IER, or the Dsg CARsequences INQ, INR and LNK.

Using linkers, claudin CAR sequence-containing peptides and otherpeptide or protein sequences may be joined head-to-tail (i.e., thelinker may be covalently attached to the carboxyl or amino group of eachpeptide sequence), head-to-side chain and/or tail-to-side chain.Modulating agents comprising one or more linkers may form linear orbranched structures. Within one embodiment, modulating agents having abranched structure may comprise a claudin CAR sequence and an occludinCAR sequence. Alternatively, a branched structure may comprise threedifferent CAR sequences, such as RGD, a claudin CAR sequence and HAV.Within another embodiment, modulating agents having a branched structurecomprise a claudin CAR sequence, along with one or more of HAV, ROD,LYHY (SEQ ID NO:17), DAE, DPK, IEK, VER, IER, INQ, INR and/or LNK.

Other combinations of CAR sequences are also possible. Linkerspreferably produce a distance between CAR sequences between 0.1 to10,000 nm, more preferably about 0.1-400 nm. A separation distancebetween recognition sites may generally be determined according to thedesired function of the modulating agent, as discussed above.

The total number of CAR sequences (including claudin CAR sequence(s)),with or without other CAR sequences derived from one or more adhesionmolecules) present within a modulating agent may range from 1 to a largenumber, such as 100, preferably from 1 to 10, and more preferably from 1to 5. Peptide modulating agents comprising multiple CAR sequencestypically contain from 6 to about 1000 amino acid residues, preferablyfrom 6 to 50 residues. When non-peptide linkers are employed, each CARsequence of the modulating agent is present within a peptide thatgenerally ranges in size from 4 to 50 residues in length, preferablyfrom 4 to 25 residues, more preferably from 4 to 16 residues and stillmore preferably from 4 to 15 residues.

As noted above, modulating agents may be polypeptides or salts thereof,containing only amino acid residues linked by peptide bonds, or maycontain non-peptide regions, such as linkers. Peptide regions of amodulating agent may comprise residues of L-amino acids, D-amino acids,or any combination thereof. Amino acids may be from natural ornon-natural sources, provided that at least one amino group and at leastone carboxyl group are present in the molecule; α- and β-amino acids aregenerally preferred. The 20 L-amino acids commonly found in proteins areidentified herein by the conventional three-letter or one-letterabbreviations, and the corresponding D-amino acids are designated by alower case one letter symbol.

A modulating agent may also contain rare amino acids (such as4-hydroxyproline or hydroxylysine), organic acids or amides and/orderivatives of common amino acids, such as amino acids having theC-terminal carboxylate esterified (e.g., benzyl, methyl or ethyl ester)or amidated and/or having modifications of the N-terminal amino group(e.g., acetylation or alkoxycarbonylation), with or without any of awide variety of side-chain modifications and/or substitutions (e.g.,methylation, benzylation, t-butylation, tosylation, alkoxycarbonylation,and the like). Preferred derivatives include amino acids having aC-terminal amide group. Residues other than common amino acids that maybe present with a modulating agent include, but are not limited to,2-mercaptoaniline, 2-mercaptoproline, ornithine, diaminobutyric acid,α-aminoadipic acid, m-aminomethylbenzoic acid and α,β-diaminopropionicacid.

Peptide modulating agents (and peptide portions of modulating agents) asdescribed herein may be synthesized by methods well known in the art,including chemical synthesis and recombinant DNA methods. For modulatingagents up to about 50 residues in length, chemical synthesis may beperformed using solution or solid phase peptide synthesis techniques, inwhich a peptide linkage occurs through the direct condensation of theα-amino group of one amino acid with the α-carboxy group of the otheramino acid with the elimination of a water molecule. Peptide bondsynthesis by direct condensation, as formulated above, requiressuppression of the reactive character of the amino group of the firstand of the carboxyl group of the second amino acid. The maskingsubstituents must permit their ready removal, without inducing breakdownof the labile peptide molecule.

In solution phase synthesis, a wide variety of coupling methods andprotecting groups may be used (see Gross and Meienhofer, eds., “ThePeptides: Analysis, Synthesis, Biology,” Vol. 1-4 (Academic Press,1979); Bodansky and Bodansky, “The Practice of Peptide Synthesis,” 2ded. (Springer Verlag, 1994)). In addition, intermediate purification andlinear scale up are possible. Those of ordinary skill in the art willappreciate that solution synthesis requires consideration of main chainand side chain protecting groups and activation method. In addition,careful segment selection is necessary to minimize racemization duringsegment condensation. Solubility considerations are also a factor.

Solid phase peptide synthesis uses an insoluble polymer for supportduring organic synthesis. The polymer-supported peptide chain permitsthe use of simple washing and filtration steps instead of laboriouspurifications at intermediate steps. Solid-phase peptide synthesis maygenerally be performed according to the method of Merrifield et al., J.Am. Chem. Soc. 85:2149, 1963, which involves assembling a linear peptidechain on a resin support using protected amino acids. Solid phasepeptide synthesis typically utilizes either the Boc or Fmoc strategy.The Boc strategy uses a 1% cross-linked polystyrene resin. The standardprotecting group for (α-amino functions is the tert-butyloxycarbonyl(Boc) group. This group can be removed with dilute solutions of strongacids such as 25% trifluoroacetic acid (TFA). The next Boc-amino acid istypically coupled to the amino acyl resin using dicyclohexylcarbodiimide(DCC). Following completion of the assembly, the peptide-resin istreated with anhydrous HF to cleave the benzyl ester link and liberatethe free peptide. Side-chain functional groups are usually blockedduring synthesis by benzyl-derived blocking groups, which are alsocleaved by HF. The free peptide is then extracted from the resin with asuitable solvent, purified and characterized. Newly synthesized peptidescan be purified, for example, by gel filtration, HPLC, partitionchromatography and/or ion-exchange chromatography, and may becharacterized by, for example, mass spectrometry or amino acid sequenceanalysis. In the Boc strategy, C-terminal amidated peptides can beobtained using benzhydrylamine or methylbenzhydrylamine resins, whichyield peptide amides directly upon cleavage with HF.

In the procedures discussed above, the selectivity of the side-chainblocking groups and of the peptide-resin link depends upon thedifferences in the rate of acidolytic cleavage. Orthoganol systems havebeen introduced in which the side-chain blocking groups and thepeptide-resin link are completely stable to the reagent used to removethe α-protecting group at each step of the synthesis. The most common ofthese methods involves the 9-fluorenylmethyloxycarbonyl (Fmoc) approach.Within this method, the side-chain protecting groups and thepeptide-resin link are completely stable to the secondary amines usedfor cleaving the N-α-Fmoc group. The side-chain protection and thepeptide-resin link are cleaved by mild acidolysis. The repeated contactwith base makes the Merrifield resin unsuitable for Fmoc chemistry, andp-alkoxybenzyl esters linked to the resin are generally used.Deprotection and cleavage are generally accomplished using TFA.

Those of ordinary skill in the art will recognize that, in solid phasesynthesis, deprotection and coupling reactions must go to completion andthe side-chain blocking groups must be stable throughout the entiresynthesis. In addition, solid phase synthesis is generally most suitablewhen peptides are to be made on a small scale.

Acetylation of the N-terminus can be accomplished by reacting the finalpeptide with acetic anhydride before cleavage from the resin.C-amidation is accomplished using an appropriate resin such asmethylbenzhydrylamine resin using the Boc technology.

Following synthesis of a linear peptide, with or without N-acetylationand/or C-amidation, cyclization may be achieved if desired by any of avariety of techniques well known in the art. Within one embodiment, abond may be generated between reactive amino acid side chains. Forexample, a disulfide bridge may be formed from a linear peptidecomprising two thiol-containing residues by oxidizing the peptide usingany of a variety of methods. Within one such method, air oxidation ofthiols can generate disulfide linkages over a period of several daysusing either basic or neutral aqueous media. The peptide is used in highdilution to minimize aggregation and intermolecular side reactions. Thismethod suffers from the disadvantage of being slow but has the advantageof only producing H₂O as a side product. Alternatively, strong oxidizingagents such as I₂ and K₃Fe(CN)₆ can be used to form disulfide linkages.Those of ordinary skill in the art will recognize that care must betaken not to oxidize the sensitive side chains of Met, Tyr, Trp or His.Cyclic peptides produced by this method require purification usingstandard techniques, but this oxidation is applicable at acid pHs.Oxidizing agents also allow concurrent deprotection/oxidation ofsuitable S-protected linear precursors to avoid premature, nonspecificoxidation of free cysteine.

DMSO, unlike I₂ and K₃Fe(CN)₆, is a mild oxidizing agent which does notcause oxidative side reactions of the nucleophilic amino acids mentionedabove. DMSO is miscible with H₂O at all concentrations, and oxidationscan be performed at acidic to neutral pHs with harmless byproducts.Methyltrichlorosilane-diphenylsulfoxide may alternatively be used as anoxidizing agent, for concurrent deprotection/oxidation of S-Acm, S-Tacmor S-t-Bu of cysteine without affecting other nucleophilic amino acids.There are no polymeric products resulting from intermolecular disulfidebond formation. Suitable thiol-containing residues for use in suchoxidation methods include, but are not limited to, cysteine,β,β-dimethyl cysteine (penicillamine or Pen), β,β-tetrarnethylenecysteine (Tmc), β,β-pentamethylene cysteine (Pmc), β-mercaptopropionicacid (Mpr), β,β-pentamethylene-β-mercaptopropionic acid (Pmp),2-mercaptobenzene, 2-mercaptoaniline and 2-mercaptoproline. Peptidescontaining such residues are illustrated by the following representativeformulas, in which the underlined portion is cyclized, N-acetyl groupsare indicated by N—Ac and C-terminal amide groups are represented by—NH₂:

i) N—Ac-Cys-Ile-Tyr-Ser-Tyr-Cys-NH₂ (SEQ ID NO:59)

ii) H-Cvs-Ile-Tyr-Ser-Tyr-Cys-OH (SEQ ID NO:59)

iii) N—Ac-Cys-Trp-Lys-Ile-Tyr-Ser-Tyr-Cys-NH₂ (SEQ ID NO:65)

iv) H-Cys-Trp-Lys-Ile-Tyr-Ser-Tyr-Cys-OH (SEQ ID NO:65)

v) N—Ac-Cys-Lys-Ile-Tyr-Ser-Tyr-Cys-NH₂ (SEQ ID NO:62)

vi) H-Cys-Lys-Ile-Tyr-Ser-Tyr-Cys-OH (SEQ ID NO:62)

vii) N—Ac-Cys-Ile-Tyr-Ser-Tyr-Pen-NH₂ (SEQ ID NO:18)

viii) N—Ac-Tmc-Ile-Tyr-Ser-Tyr-Cys-NH₂ (SEQ ID NO:19)

ix) N—Ac-Pmc-Ile-Tyr-Ser-Tyr-Cys-NH₂ (SEQ ID NO:20)

x) N—Ac-Mpr-Ile-Tyr-Ser-Tyr-Cys-NH₂ (SEQ ID NO:21)

xi) N—Ac-Pmp-Ile-Tyr-Ser-Tyr-Cys-NH₂ (SEQ ID NO:22)

It will be readily apparent to those of ordinary skill in the art that,within each of these representative formulas, any of the abovethiol-containing residues may be employed in place of one or both of thethiol-containing residues recited.

Within another embodiment, cyclization may be achieved by amide bondformation. For example, a peptide bond may be formed between terminalfunctional groups (i.e., the amino and carboxy termini of a linearpeptide prior to cyclization), as in KIYSY (SEQ ID NO:106). Withinanother such embodiment, the cyclic peptide comprises a D-amino acid(e.g., yIYSY; SEQ ID NO:23). Alternatively, cyclization may beaccomplished by linking one terminus and a residue side chain or usingtwo side chains, as in KIYSYD (SEQ ID NO:68), with or without anN-terminal acetyl group and/or a C-terminal amide. Residues capable offorming a lactam bond include lysine, ornithine (Orn), α-amino adipicacid, m-aminomethylbenzoic acid, α,β-diaminopropionic acid, glutamate oraspartate.

Methods for forming amide bonds are well known in the art and are basedon well established principles of chemical reactivity. Within one suchmethod, carbodiimide-mediated lactam formation can be accomplished byreaction of the carboxylic acid with DCC, DIC, EDAC or DCCI, resultingin the formation of an O-acylurea that can be reacted immediately withthe free amino group to complete the cyclization. The formation of theinactive N-acylurea, resulting from O→N migration, can be circumventedby converting the O-acylurea to an active ester by reaction with anN-hydroxy compound such as 1-hydroxybenzotriazole, 1-hydroxysuccinimide,1-hydroxynorbornene carboxamide or ethyl 2-hydroximino-2-cyanoacetate.In addition to minimizing O→N migration, these additives also serve ascatalysts during cyclization and assist in lowering racemization.Alternatively, cyclization can be performed using the azide method, inwhich a reactive azide intermediate is generated from an alkyl ester viaa hydrazide. Hydrazinolysis of the terminal ester necessitates the useof a t-butyl group for the protection of side chain carboxyl functionsin the acylating component. This limitation can be overcome by usingdiphenylphosphoryl acid (DPPA), which furnishes an azide directly uponreaction with a carboxyl group. The slow reactivity of azides and theformation of isocyanates by their disproportionation restrict theusefulness of this method. The mixed anhydride method of lactamformation is widely used because of the facile removal of reactionby-products. The anhydride is formed upon reaction of the carboxylateanion with an alkyl chloroformate or pivaloyl chloride. The attack ofthe amino component is then guided to the carbonyl carbon of theacylating component by the electron donating effect of the alkoxy groupor by the steric bulk of the pivaloyl chloride t-butyl group, whichobstructs attack on the wrong carbonyl group. Mixed anhydrides withphosphoric acid derivatives have also been successfully used.Alternatively, cyclization can be accomplished using activated esters.The presence of electron withdrawing substituents on the alkoxy carbonof esters increases their susceptibility to aminolysis. The highreactivity of esters of p-nitrophenol, N-hydroxy compounds andpolyhalogenated phenols has made these “active esters” useful in thesynthesis of amide bonds. The last few years have witnessed thedevelopment of benzotriazolyloxytris(dimethylamino)phosphoniumhexafluorophosphonate (BOP) and its congeners as advantageous couplingreagents. Their performance is generally superior to that of the wellestablished carbodiimide amide bond formation reactions.

Within a further embodiment, a thioether linkage may be formed betweenthe side chain of a thiol-containing residue and an appropriatelyderivatized α-amino acid. By way of example, a lysine side chain can becoupled to bromoacetic acid through the carbodiimide coupling method(DCC, EDAC) and then reacted with the side chain of any of the thiolcontaining residues mentioned above to form a thioether linkage. Inorder to form dithioethers, any two thiol containing side-chains can bereacted with dibromoethane and diisopropylamine in DMF. Examples ofthiol-containing linkages are shown below:

Cyclization may also be achieved using δ₁,δ₁-Ditryptophan (i.e.,Ac-Trp-Gly-Gly-Trp-OMe) (SEQ ID NO:24), as shown below:

Representative structures of cyclic peptides are provided in FIGS. 2Aand 2B. The structures and formulas recited herein are provided solelyfor the purpose of illustration, and are not intended to limit the scopeof the cyclic peptides described herein.

For longer modulating agents, recombinant methods are preferred forsynthesis. Within such methods, all or part of a modulating agent can besynthesized in living cells, using any of a variety of expressionvectors known to those of ordinary skill in the art to be appropriatefor the particular host cell. Suitable host cells may include bacteria,yeast cells, mammalian cells, insect cells, plant cells, algae and otheranimal cells (e.g., hybridoma, CHO, myeloma). The DNA sequencesexpressed in this manner may encode portions of an endogenous claudin orother adhesion molecule. Such sequences may be prepared based on knowncDNA or genomic sequences, or from sequences isolated by screening anappropriate library with probes designed based on known claudinsequences. Such screens may generally be performed as described inSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratories, Cold Spring Harbor, N.Y., 1989 (and referencescited therein). Polymerase chain reaction (PCR) may also be employed,using oligonucleotide primers in methods well known in the art, toisolate nucleic acid molecules encoding all or a portion of anendogenous adhesion molecule. To generate a nucleic acid moleculeencoding a desired modulating agent, an endogenous claudin sequence maybe modified using well known techniques. For example, portions encodingone or more CAR sequences may be joined, with or without separation bynucleic acid regions encoding linkers, as discussed above.Alternatively, portions of the desired nucleic acid sequences may besynthesized using well known techniques, and then ligated together toform a sequence encoding the modulating agent.

As noted above, polynucleotides may also function as modulating agents.In general, such polynucleotides should be formulated to permitexpression of a polypeptide modulating agent following administration toa mammal. Such formulations are particularly useful for therapeuticpurposes, as described below. Those of ordinary skill in the art willappreciate that there are many ways to achieve expression of apolynucleotide within a mammal, and any suitable method may be employed.For example, a polynucleotide may be incorporated into a viral vectorsuch as, but not limited to, adenovirus, adeno-associated virus,retrovirus, or vaccinia or other pox virus (e.g., avian pox virus).Techniques for incorporating DNA into such vectors are well known tothose of ordinary skill in the art. A retroviral vector may additionallytransfer or incorporate a gene for a selectable marker (to aid in theidentification or selection of transfected cells) and/or a targetingmoiety, such as a gene that encodes a ligand for a receptor on aspecific target cell, to render the vector target specific. Targetingmay also be accomplished using an antibody, by methods known to those ofordinary skill in the art. Other formulations for polynucleotides fortherapeutic purposes include colloidal dispersion systems, such asmacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. A preferred colloidal system for use as adelivery vehicle in vitro and in vivo is a liposome (i.e., an artificialmembrane vesicle). The preparation and use of such systems is well knownin the art.

As noted above, instead of (or in addition to) a claudin CAR sequence, amodulating agent may comprise an antibody, or antigen-binding fragmentthereof, that specifically binds to a claudin CAR sequence. As usedherein, an antibody, or antigen-binding fragment thereof, is said to“specifically bind” to a claudin CAR sequence (with or without flankingamino acids) if it reacts at a detectable level with a peptidecontaining that sequence, and does not react detectably with peptidescontaining a different CAR sequence or a sequence in which the order ofamino acid residues in the claudin CAR sequence and/or flanking sequenceis altered. Such antibody binding properties may be assessed using anELISA, as described by Newton et al., Develop. Dynamics 197:1-13, 1993.

Polyclonal and monoclonal antibodies may be raised against a claudin CARsequence using conventional techniques. See, e.g., Harlow and Lane,Antibodies: A Laboralory Manual, Cold Spring Harbor Laboratory, 1988. Inone such technique, an immunogen comprising the claudin CAR sequence isinitially injected into any of a wide variety of mammals (e.g., mice,rats, rabbits, sheep or goats). The smaller immunogens (i.e., less thanabout 20 amino acids) should be joined to a carrier protein, such asbovine serum albumin or keyhole limpet hemocyanin. Following one or moreinjections, the animals are bled periodically. Polyclonal antibodiesspecific for the CAR sequence may then be purified from such antiseraby, for example, affinity chromatography using the modulating agent orantigenic portion thereof coupled to a suitable solid support.

Monoclonal antibodies specific for the claudin CAR sequence may beprepared, for example, using the technique of Kohler and Milstein, Eur.J. Immunol. 6:511-519, 1976, and improvements thereto. Briefly, thesemethods involve the preparation of immortal cell lines capable ofproducing antibodies having the desired specificity from spleen cellsobtained from an animal immunized as described above. The spleen cellsare immortalized by, for example, fusion with a myeloma cell fusionpartner, preferably one that is syngeneic with the immunized animal.Single colonies are selected and their culture supernatants tested forbinding activity against the modulating agent or antigenic portionthereof. Hybridomas having high reactivity and specificity arepreferred.

Monoclonal antibodies may be isolated from the supernatants of growinghybridoma colonies, with or without the use of various techniques knownin the art to enhance the yield. Contaminants may be removed from theantibodies by conventional techniques, such as chromatography, gelfiltration, precipitation, and extraction. Antibodies having the desiredactivity may generally be identified using immunofluorescence analysesof tissue sections, cell or other samples where the target claudin islocalized.

Within certain embodiments, the use of antigen-binding fragments ofantibodies may be preferred. Such fragments include Fab fragments, whichmay be prepared using standard techniques. Briefly, immunoglobulins maybe purified from rabbit serum by affinity chromatography on Protein Abead columns (Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, 1988, see especially page 309) and digested bypapain to yield Fab and Fc fragments. The Fab and Fc fragments may beseparated by affinity chromatography on protein A bead columns (Harlowand Lane, 1988, pages 628-29).

Evaluation of Modulating Agent Activity

As noted above, modulating agents as described herein are capable ofmodulating claudin-mediated cell adhesion. The ability of an agent tomodulate cell adhesion may generally be evaluated in vitro by assayingthe effect on endothelial and/or epithelial cell adhesion or cellstransfected with claudin cDNA, such as L cells (Furuse et al. J CellBiol. 143:391-401, 1998), using, for example, any of a variety ofimmunostaining protocols and/or plating assays. In general, a modulatingagent is an inhibitor of cell adhesion if contact of the test cells withthe modulating agent results in a discernible disruption of celladhesion using one or more representative assays provided herein.Modulating agents that enhance cell adhesion (e.g., agents comprisingmultiple claudin CAR sequences and/or linked to a support molecule ormaterial) are considered to be modulators of cell adhesion if they arecapable of promoting cell adhesion, as judged by plating assays toassess either endothelial or epithelial cell adhesion to a modulatingagent attached to a support material, such as tissue culture plastic.

An initial screen for the ability to modulate one or moreclaudin-mediated functions may be performed by evaluating the ability ofa modulating agent to bind to a claudin using any binding assay known tothose of ordinary skill in the art. For example, a Pharmacia Biosensormachine may be used, as discussed in Jonsson et al., Biotechniques11:520-27, 1991. For example, a modulating agent may comprise a CARsequence that binds to a claudin. A specific example of a technologythat measures the interaction of peptides with molecules can be found inWilliams et al., J Biol. Chem. 272, 22349-22354, 1997. Alternatively,real-time BIA (Biomolecular Interaction Analysis) uses the opticalphenomenon surface plasmon resonance to monitor biomolecularinteractions. The detection depends upon changes in the massconcentration of macromolecules at the biospecific interface, which inturn depends upon the immobilization of test molecule or peptide(referred to as the ligand) to the surface of a Biosensor chip, followedby binding of the interacting molecule (referred to as the analyte) tothe ligand. Binding to the chip is measured in real-time in arbitraryunits of resonance (RU).

By way of example, surface plasmon resonance experiments may be carriedout using a BIAcore X™ Biosensor (Pharmacia Ltd., BIAcore, Uppsala,Sweden). Parallel flow cells of CM 5 sensor chips may be derivatized,using the amine. coupling method, with sireptavidin (200 μg/ml) in 10 mMSodium Acetate, pH 4.0, according to the manufacturer's protocol.Approximately 2100-2600 resonance units (RU) of ligand may beimmobilized, corresponding to a concentration of about 2.1-2.6 ng/mm².The chips may then coated be with claudin derivatized to biotin. Anynon-specifically bound protein is removed.

To determine binding, test analytes (e.g., peptides containing theclaudin CAR sequence) may be placed in running buffer and passedsimultaneously over test and control flow cells. After a period of freebuffer flow, any analyte remaining bound to the surface may be removedwith, for example, a pulse of 0.1% SDS bringing the signal back tobaseline. Specific binding to the derivatized sensor chips may bedetermined automatically by the system by subtraction of test fromcontrol flow cell responses. In general, a modulating agent binds to aclaudin at a detectable level within such as assay. The level of bindingis preferably at least that observed for the full length claudin undersimilar conditions.

The ability of an agent to modulate cell adhesion may generally beevaluated in vivo by assessing the effect on vascular permeabilityutilizing the Miles assay (McClure et al., J. Pharmacological &Toxicological Methods 32:49-52, 1994). Briefly, a candidate modulatingagent may be dissolved in phosphate buffered saline (PBS) at aconcentration of 100 μg/ml. Adult rats may be given 100 μl subdermalinjections of each peptide solution into their shaved backs, followed 15minutes later by a single 250 μl injection of 1% Evans blue dissolved inPBS into their tail veins. The subdermal injection sites may be visuallymonitored for the appearance of blue dye. Once the dye appears (about 15minutes after injection), each subdermal injection site may be excised,weighed, and placed in 1 ml dimethylformamide for 24 hours to extractthe dye. The optical density of the dye extracts may then be determinedat 620 nm. In general, the injection of 0.1 ml of modulating agent (at aconcentration of 0.1 mg/ml) into the backs of rats causes an increase ofdye accumulation at the injection sites of at least 50%, as compared todye accumulation at sites into which PBS has been injected.

The effect of a modulating agent on endothelial cell adhesion maygenerally be evaluated using immunolocalization techniques. Human aorticendothelial cells (HAEC) may be cultured on fibronectin-coatedcoverslips (fibronectin may be obtained from Sigma, St. Louis, Mo.)according to the procedures of Jaffe. et al., J. Clin. Invest.52:2745-2756, 1973. Briefly, human endothelial cells may be maintainedin EGM (endothelial cell growth medium; Clonetics, San Diego: Calif.)and used for experiments at passage 4. Confluent cultures of HAEC may beexposed to either a candidate modulating agent (final concentration 100μg/ml EGM), or EGM alone for 1 hour. The cells are then be fixed for 30minutes at 4° C. in 95% ethanol, followed by fixation in acetone for 1minute at 4° C. (Furuse et al., J. Cell Biol. 123:1777-1788, 1993).After fixation, the cells may be probed with either mouseanti-VE-cadherin antibodies (Hemeris, Sassenage, France; diluted 1:250in 0.1% dried skim milk powder dissolved in PBS), or anti-claudinantibodies (prepared as described by Morita et al., Proc. Natl. AcadSci. USA 96:511-516, 1999) diluted in 0.1% dried skim milk powderdissolved in PBS) for 1 hour at 37° C. The cells may then be washed with0.1% dried skim milk powder dissolved in PBS (three washes, 5minutes/wash), and probed with secondary antibodies (donkey anti-mouseCy3, or donkey anti-rabbit Cy5 diluted 1:250 in 0.1% dried skim milkpowder dissolved in PBS; Jackson Immunoresearch Laboratories Inc.,Westgrove, Pa.) for 1 hour at 37° C. The cells may then be washed againwith in 0.1% dried skim milk powder dissolved in PBS and mounted in asolution composed of 50% glycerol and 50% PBS to which phenylenediamine(Sigma, St. Louis, Mo.) has been added to a final concentration of 1mg/ml. The sample, may then be analyzed using a Bio-Rad MRC 1000confocal microscope with Laser Sharp software version 2.1T (Bio-Rad,Hercules, Calif.). In general, 0.1 mg/ml of modulating agent results inthe appearance of intercellular gaps within the monolayer cultures and adecrease of at least 50% in the surface expression of claudin andVE-cadherin, as compared to monolayer cultures that were not exposed tothe modulating agent.

Within certain cell adhesion assays, the addition of a modulating agentto cells that express claudin results in disruption of cell adhesion. A“claudin-expressing cell,” as used herein, may be any type of cell thatexpresses claudin on the cell surface at a detectable level, usingstandard techniques such as immunocytochemical protocols (e.g., Blaschukand Farookhi, Dev. Biol. 136:564-567, 1989). Claudin-expressing cellsinclude endothelial, epithelial and/or cancer cells. For example, suchcells may be plated under standard conditions that, in the absence ofmodulating agent, permit cell adhesion. In the presence of modulatingagent (e.g., 100 μg/mL), disruption of cell adhesion may be determinedvisually within 24 hours, by observing retraction of the cells from oneanother.

Within another such assay, the effect of a modulating agent on normalrat kidney (NRK) cells may be evaluated. According to a representativeprocedure, NRK cells (ATCC #1571-CRL) may be plated at 10-20,000 cellsper 35 mm tissue culture flasks containing DMEM with 10% FCS andsub-cultured periodically (Laird et al., J. Cell Biol. 131:1193-1203,1995). Cells may be harvested and replated in 35 mm tissue cultureflasks containing 1 mm coverslips and incubated until 50-65% confluent(24-36 hours). At this time, coverslips may be transferred to a 24-wellplate, washed once with fresh DMEM and exposed to modulating agent at aconcentration of, for example, 0.1 mg/mL for 24 hours. Fresh modulatingagent may then be added, and the cells left for an additional 24 hours.Cells may be fixed with 100% methanol for 10 minutes and then washedthree times with PBS. Coverslips may be blocked for 1 hour in 2% BSA/PBSand incubated for a further 1 hour in the presence of anti-claudinantibody and mouse anti-E-cadherin antibody (Transduction Labs, 1:250dilution). Primary and secondary antibodies may be diluted in 2%BSA!PBS. Following incubation in the primary antibody, coverslips may bewashed three times for 5 minutes each in PBS and incubated for 1 hourwith donkey anti-mouse Cy3 and donkey anti-rabbit Cy5 (JacksonImmunoresearch Laboratories Inc., Westgrove, Pa.) for 1 hour at 37° C.Following further washes in PBS (3×5 min) coverslips can be mounted andviewed by confocal microscopy.

In the absence of modulating agent, NRK cells form characteristictightly adherent monolayers with a cobblestone morphology in which cellsdisplay a polygonal shape. NRK cells that are treated with a modulatingagent that disrupts claudin-mediated cell adhesion may assume anon-polygonal and elongated morphology (i.e., a fibroblast-like shape)within 48 hours of treatment with 0.1 mg/mL of modulating agent. Gapsappear in confluent cultures of such cells. In addition, 0.1 mg/mL ofsuch a modulating agent reproducibly induces a readily apparentreduction in cell surface staining of claudin and E-cadherin, as judgedby immunofluorescence microscopy (Laird et al., J. Cell Biol.131:1193-1203, 1995), preferably at least 75% within 48 hours.

A third cell adhesion assay involves evaluating the effect of amodulating agent on permeability of adherent endothelial cellmonolayers. The effects of a modulating agent on the permeability ofendothelial cell monolayers may be assessed utilizing the protocols ofEhringer et al., J. Cell. Physiol. 167:562-569, 1996. HAEC can be seededonto inserts in 24-well plates (Becton-Dickenson, Franklin Lake, N.J.)and cultured in EGM. Confluent cell monolayers may be exposed to eithermodulating agent (final concentration 100 μg/ml EGM), or EGM alone for 1hour. The inserts may then be transferred to 24-chamber plates(Becton-Dickenson) for permeability assays. Perfusate (0.5% bovine serumalbumin, fraction V (Sigma) dissolved in 15 mM HEPES, pH 7.4) andFITC-Dextran (50 μg/ml HEPES buffer; MW 12 kDa; Sigma) may be added toeach well (1 ml/well and 50 μl/well, respectively), and the cellsincubated at 37° C. for 30 min. Aliquots of 100 μl may then be removedfrom the lower chamber and the optical density of the solutiondetermined at a wavelength of 450 nm. In general, the presence of 100μg/mL modulating agent that enhances the permeability of endothelialcell monolayers results in a statistically significant increase in theamount of marker in the receptor compartment after 1 hour.

Alternatively, cells that do not naturally express a claudin may be usedwithin such assays. Such cells may be stably transfected with apolynucleotide (e.g., a cDNA) encoding a claudin of interest, such thatthe claudin is expressed on the surface of the cell. Transfection ofcells for use in cell adhesion assays may be performed using standardtechniques and published claudin sequences. Expression of the claudinmay be confirmed by assessing adhesion of the transfected cells, inconjunction with immunocytochemical techniques using antibodies directedagainst the claudin of interest. The stably transfected cells thataggregate, as judged by light microscopy, following transfection expresssufficient levels of the claudin. Preferred cells for use in such assaysinclude L cells, which do not delectably adhere in the absence oftransfection (Nagafuchi et al., Nature 329:341-343, 1987). Followingtransfection of L cells with a cDNA encoding a claudin, cell adhesionmay be observed (Furuse et al J Cell Biol. 143:391-401, 1998).Modulating agents that detectably inhibit such aggregation may be usedto modulate functions mediated by the claudin. Such assays have beenused for numerous nonclassical cadherins, including OB-cadherin (Okazakiet al., J. Biol. Chem. 269:12092-98, 1994), cadherin-5 (Breier et al.,Blood 87:630-641, 1996), cadherin-6 (Mbalaviele et al., J. Cell. Biol.141:1467-1476, 1998), cadherin-8 (Kido et al., Genomics 48:186-194,1998), cadherin-15 (Shimoyama et al., J. Biol. Chem. 273:10011-10018,1998), PB-cadherin (Sugimoto et al., J. Biol. Chem. 271:11548-11556,1996), LI-cadherin (Kreft et al., J. Cell. Biol. 136:1109-1121, 1997),protocadherin 42 and 43 (Sano et al., EMBO J. 12:2249-2256, 1993) anddesmosomal cadherins (Marcozzi et al., J. Cell. Sci. 111:495-509, 1998).It will be apparent to those of ordinary skill in the art that assaysmay be performed in a similar manner for claudins. In general, amodulating agent that is derived from a particular claudin CAR sequence(i.e., comprises such a CAR sequence, or an analog or mimetic thereof,or an antibody that specifically recognizes such a CAR sequence) andthat modulates adhesion of a cell that expresses the same claudin isconsidered to modulate a function mediated by the claudin.

Yet another assay evaluates the effect of a modulating agent on theelectrical resistance across a monolayer of cells. For example, MadinDarby canine kidney (MDCK) cells can be exposed to the modulating agentdissolved in medium (e.g., at a final concentration of 0.5 mg/ml for aperiod of 24 hours). The effect on electrical resistance can be measuredusing standard techniques. This assay evaluates the effect of amodulating agent on tight junction formation in epithelial cells. Ingeneral, the presence of 500 μg/mL modulating agent should result in astatistically significant decrease in electrical resistance after 24hours.

Modulating Agent Modification and Formulation

A modulating agent as described herein may, but need not, be linked toone or more additional molecules. In particular, as discussed below, itmay, be beneficial for certain applications to link multiple modulatingagents (which may, but need not, be identical) to a support material,such as a single molecule (e.g., keyhole limpet hemocyanin) or a solidsupport, such as a polymeric matrix (which may be formulated as amembrane or microstructure, such as an ultra thin film), a containersurface (e.g., the surface of a tissue culture plate or the interiorsurface of a bioreactor), or a bead or other particle, which may beprepared from a variety of materials including glass, plastic orceramics. For certain applications, biodegradable support materials arepreferred, such as cellulose and derivatives thereof, collagen, spidersilk or any of a variety of polyesters (e.g., those derived from hydroxyacids and/ore lactones) or sutures (see U.S. Pat. No. 5,245,012). Withincertain embodiments, modulating agents and molecules comprising otherCAR sequence(s) (e.g., an HAV sequence) may be attached to a supportsuch as a polymeric matrix, preferably in an alternating, pattern.

Suitable methods for linking a modulating agent to a support materialwill depend upon the composition of the support and the intended use,and will be readily apparent to those of ordinary skill in the art.Attachment may generally be achieved through noncovalent association,such as adsorption or affinity or, preferably, via covalent attachment(which may be a direct linkage between a modulating agent and functionalgroups on the support, or may be a linkage by way of a cross-linkingagent). Attachment of a modulating agent by adsorption may be achievedby contact, in a suitable buffer, with a solid support for a suitableamount of time. The contact time varies with temperature, but isgenerally between about 5 seconds and 1 day, and typically between about10 seconds and 1 hour.

Covalent attachment of a modulating agent to a molecule or solid supportmay generally be achieved by first reacting the support material with abifunctional reagent that will also react with a functional group, suchas a hydroxyl, thiol, carboxyl, ketone or amino group, on the modulatingagent. For example, a modulating agent may be bound to an appropriatepolymeric support or coating using benzoquinone, by condensation of analdehyde group on the support with an amine and an active hydrogen onthe modulating agent or by condensation of an amino group on the supportwith a carboxylic acid on the modulating agent. A preferred method ofgenerating a linkage is via amino groups using glutaraldehyde. Amodulating agent may be linked to cellulose via ester linkages.Similarly, amide linkages may be suitable for linkage to other moleculessuch as keyhole limpet hemocyanin or other support materials. Multiplemodulating agents and/or molecules comprising other CAR sequences may beattached, for example, by random coupling, in which equimolar amounts ofsuch molecules are mixed with a matrix support and allowed to couple atrandom.

Although modulating agents as described herein may preferentially bindto specific tissues or cells, and thus may be sufficient to target adesired site in vivo, it may be beneficial for certain applications toinclude an additional targeting agent. Accordingly, a targeting agentmay also, or alternatively, be linked to a modulating agent tofacilitate targeting to one or more specific tissues. As used herein, a“targeting agent,” may be any substance (such as a compound or cell)that, when linked to a modulating agent enhances the transport of themodulating agent to a target tissue, thereby increasing the localconcentration of the modulating agent. Targeting agents includeantibodies or fragments thereof, receptors, ligands and other moleculesthat bind to cells of, or in the vicinity of, the target tissue. Knowntargeting agents include serum hormones, antibodies against cell surfaceantigens, lectins, adhesion molecules, tumor cell surface bindingligands, steroids, cholesterol, lymphokines, fibrinolytic enzymes andthose drugs and proteins that bind to a desired target site. Forexample, in receptor-mediated delivery, a modulating agent may be linkedto a ligand that recognizes a specific receptor on the surface of atarget cell. In certain instances, modulating agent is released withinthe cell following cleavage with intracellular enzymes. Among the manymonoclonal antibodies that may serve as targeting agents are anti-TAC,or other interleukin-2 receptor antibodies; 9.2.27 and NR-ML-05,reactive with the 250 kilodalton human melanoma-associated proieoglycan;and NR-LU-10, reactive with a pancarcinoma glycoprotein. An antibodytargeting agent may be an intact (whole) molecule, a fragment thereof,or a functional equivalent thereof. Examples of antibody fragments areF(ab′)2, -Fab′, Fab and F[v] fragments, which may be produced byconventional methods or by genetic or protein engineering. Linkage isgenerally covalent and may be achieved by, for example, directcondensation or other reactions, or by way of bi- or multi-functionallinkers.

For certain embodiments, it may be beneficial to also, or alternatively,link a drug to a modulating agent. As used herein, the term “drug”refers to any bioactive agent intended for administration to a mammal toprevent or treat a disease or other undesirable condition. Drugs includehormones, growth factors, proteins, peptides and other compounds. Theuse of certain specific drugs within the context of the presentinvention is discussed below.

Modulating agents as described herein may be present within apharmaceutical composition. A pharmaceutical composition comprises oneor more modulating agents in combination with one or morepharmaceutically or physiologically acceptable carriers, diluents orexcipients. Such compositions may comprise buffers (e.g., neutralbuffered saline or phosphate buffered saline), carbohydrates (e.g.,glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptidesor amino acids such as glycine, antioxidants, chelating agents such asEDTA or glutathione, adjuvants (e.g., aluminum hydroxide) and/orpreservatives. Within yet other embodiments, compositions of the presentinvention may be formulated as a lyophilizate. One or more modulatingagents (alone or in combination with a targeting agent and/or drug) may,but need not, be encapsulated within liposomes using well knowntechnology. Compositions of the present invention may be formulated forany appropriate manner of administration, including for example,topical, oral, nasal, intravenous, intracranial, intraperitoneal,subcutaneous, or intramuscular administration.

For certain embodiments, as discussed below, a pharmaceuticalcomposition may further comprise a modulator of cell adhesion that ismediated by one or more molecules other than claudin. Such modulatorsmay generally be prepared as described above, except that one or morenon-claudin CAR sequences and/or antibodies thereto are substituted forthe claudin, CAR sequence. Such compositions are particularly useful forsituations in which it is desirable to inhibit cell adhesion mediated bymultiple cell adhesion molecules, such as other members of the cadheringene superfamily (e.g., classical cadherins such as E-cadherin and/ornonclassical cadherins such as VE-cadherin, Dsg and Dsc); integrins;members of the immunoglobulin supergene family, such as N-CAM, JAM andPECAM. Preferred CAR sequences for use within such a modulator includeHAV, RGD, DDK, EEY, EAQ (OB-cads), DPK (JAM), DAE (VE-cad), IEK, VER,IER, INQ, INR and/or LNK. Also preferred is the occludin CAR sequenceLYHY (SEQ ID NO:17).

A pharmaceutical composition may also, or alternatively, contain one ormore drugs, which may be linked to a modulating agent or may be freewithin the composition. Virtually any drug may be administered incombination with a modulating agent as described herein, for a varietyof purposes as described below. Examples of types of drugs that may beadministered with a modulating agent include analgesics, anesthetics,antianginals, antifungals, antibiotics, anticancer drugs (e.g., taxol ormitomycin C), antiinflammatories (e.g., ibuprofen and indomethacin),anthelmintics, antidepressants, antidotes, antiemetics, antihistamines,antihypertensives, antimalarials, antimicrotubule agents (e.g.,colchicine or vinca alkaloids), antimigraine agents, antimicrobials,antiphsychotics, antipyretics, antiseptics, anti-signaling agents (e.g.,protein kinase C inhibitors or inhibitors of intracellular calciummobilization), antiarthritics, antithrombin agents, antituberculotics,antitussives, antivirals, appetite suppressants, cardioactive drugs,chemical dependency drugs, cathartics, chemotherapeutic agents,coronary, cerebral or peripheral vasodilators, contraceptive agents,depressants, diuretics, expectorants, growth factors, hormonal agents,hypnotics, immunosuppression agents, narcotic antagonists,parasympathomimetics, sedatives, stimulants, sympathomimetics, toxins(e.g., cholera toxin), tranquilizers and urinary antiinfectives.

For imaging purposes, any of a variety of diagnostic agents may beincorporated into a pharmaceutical composition, either linked to amodulating agent or free within the composition. Diagnostic agentsinclude any substance administered to illuminate a physiologicalfunction within a patient, while leaving other physiological functionsgenerally unaffected. Diagnostic agents include metals, radioactiveisotopes and radioopaque agents (e.g., gallium, technetium, indium,strontium, iodine, barium, bromine and phosphorus-containing compounds),radiolucent agents, contrast agents, dyes (e.g., fluorescent dyes andchromophores) and enzymes that catalyze a colorimetric or fluorometricreaction. In general, such agents may be attached using a variety oftechniques as described above, and may be present in any orientation.

The compositions described herein may be administered as part of asustained release formulation (i.e., a formulation such as a capsule orsponge that effects a slow release of modulating agent followingadministration). Such formulations may generally be prepared using wellknown technology and administered by, for example, oral, rectal orsubcutaneous implantation, or by implantation at the desired targetsite. Sustained-release formulations may contain a modulating agentdispersed in a carrier matrix and/or contained within a reservoirsurrounded by a rate controlling membrane (see, e.g., European PatentApplication 710,491 A). Carriers for use within such formulations arebiocompatible, and may also be biodegradable; preferably the formulationprovides a relatively constant level of modulating agent release. Theamount of modulating agent contained within a sustained releaseformulation depends upon the site of implantation, the rate and expectedduration of release and the nature of the condition to be treated orprevented.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented).Appropriate dosages and a suitable duration and frequency ofadministration will be determined by such factors as the condition ofthe patient, the type and severity of the patient's disease and themethod of administration. In general, an appropriate dosage andtreatment regimen provides the modulating agent(s) in an amountsufficient to provide therapeutic and/or prophylactic benefit. Withinparticularly preferred embodiments of the invention, a modulating agentor pharmaceutical composition as described herein may be administered ata dosage ranging from 0.001 to 50 mg/kg body weight, preferably from 0.1to 20 mg/kg, on a regimen of single or multiple daily doses. For topicaladministration, a cream typically comprises an amount of modulatingagent ranging from 0.00001% to 1%, preferably from 0.0001% to 0.2% andmore preferably from 0.01% to 0.1%. Fluid compositions typically containan amount of modulating agent ranging from 10 ng/ml to 5 mg/ml,preferably from 10 μg to 2 mg/mL. Appropriate dosages may generally bedetermined using experimental models and/or clinical trials. In general,the use of the minimum dosage that is sufficient to provide effectivetherapy is preferred. Patients may generally be monitored fortherapeutic effectiveness using assays suitable for the condition beingtreated or prevented, which will be familiar to those of ordinary skillin the art.

Modulating Agent Methods of Use

In general, the modulating agents and compositions described herein maybe used for modulating the adhesion of claudin-expressing cells in vitroand/or in vivo, preferably in a mammal such as a human, by contactingthe claudin-expressing cell with the modulating agent. As noted above,modulating agents for purposes that involve the disruption ofclaudin-mediated cell adhesion may comprise a claudin CAR sequence,multiple claudin CAR sequences in close proximity and/or an antibody (oran antigen-binding fragment thereof) that recognizes the claudin CARsequence. When it is desirable to also disrupt cell adhesion mediated byother adhesion molecules, a modulating agent may additionally compriseone or more CAR sequences bound by such adhesion molecules (and/orantibodies or fragments thereof that bind such sequences), preferablyseparated from each other and from the claudin CAR sequence by linkers.As noted above, such linkers may or may not comprise one or more aminoacids. For enhancing cell adhesion, a modulating agent may containmultiple claudin CAR sequences or antibodies (or fragments), preferablyseparated by linkers, and/or may be linked to a single molecule or to asupport material as described above. When it is desirable to alsoenhance cell adhesion mediated by other adhesion molecules, a modulatingagent may additionally comprise one or more CAR sequences bound by suchadhesion molecules (and/or antibodies or fragments thereof that bindsuch sequences), preferably separated from each other and from theclaudin CAR sequence by linker.

Certain methods involving the disruption of cell adhesion as describedherein have an advantage over prior techniques in that they permit thepassage of molecules that are large and/or charged across barriers ofclaudin-expressing cells. As described in greater detail below,modulating agents as described herein may also be used to disrupt orenhance cell adhesion in a variety of other contexts. Within each of themethods described herein, one or more modulating agents may generally beadministered alone, or within a pharmaceutical composition. In eachspecific method described herein, as noted above, a targeting agent maybe employed to increase the local concentration of modulating agent atthe target site.

The present invention provides, within certain aspects, methods forincreasing vasopermeability in a mammal by administering one or moremodulating agents or pharmaceutical compositions. Endothelial celladhesion may be disrupted by linear and cyclic peptides containing aclaudin CAR sequence. Within blood vessels, endothelial cell adhesionresults in decreased vascular permeability. Accordingly, modulatingagents that disrupt claudin-mediated cell adhesion as described herein,can increase vascular permeability and thus may facilitate drug deliveryto previously inaccessible tissues, such as the brain. In oneparticularly preferred embodiment, a modulating agent for use withinsuch methods is capable of disrupting cell adhesion mediated by multipleadhesion molecules. For example, a single branched modulating agent (ormultiple agents linked to a single molecule or support material) maydisrupt claudin, occludin and cadherin mediated cell adhesion, therebydisrupting tight junctions and adherens junctions. Tri-functionalmodulating agents comprising a claudin CAR sequence joined to thecadherin CAR sequence HAV, and the occludin CAR sequence LYHY,preferably by a linker, are also preferred. Alternatively, a separatemodulator of non-claudin-mediated cell adhesion may be administered inconjunction with the modulating agent(s), either within the samepharmaceutical composition or separately. Preferred antibody modulatingagents that may be used in conjunction with the claudin modulatingagents include Fab fragments directed against an N-cadherin CARsequence, such as FHLRAHAVDINGNQV-NH₂ (SEQ ID NO:25), an occludin CARsequence, such as QYLYHYCVVD-NH₂ (SEQ ID NO:478), or an OB-cadherin CARsequence, such as IFVIDDKSG-NH₂ (SEQ ID NO:479).

Within certain embodiments, preferred modulating agents for use withinsuch methods include peptides capable of decreasing both endothelial andtumor cell adhesion. Such modulating agents may be used to facilitatethe penetration of anti-tumor therapeutic or diagnostic agents (e.g.,monoclonal antibodies) through endothelial cell permeability barriersand tumor barriers. In one particularly preferred embodiment, amodulating agent is capable of disrupting cell adhesion mediated bymultiple adhesion molecules. For example, a single branched modulatingagent (or multiple agents linked to a single molecule or supportmaterial) may disrupt claudin, occludin, classical cadherin, integrin,and nonclassical cadherin (e.g., Dsc and/or Dsg) mediated cell adhesion,thereby disrupting tight junctions, adherens junctions, focal contactsand desmosomes. Multifunctional modulating agents comprising a claudinCAR sequence linked to one or more of the classical cadherin CARsequence HAV; the sequence RGD, which is bound by integrins; thesequence LYHY, which is bound by occludin, and/or a nonclassicalcadherin CAR sequence, such as a Dsc CAR sequence (IEK, VER or IER)and/or a Dsg CAR sequence (INQ, INR or LNK), may be used to disrupt celladhesion. Alternatively, a separate modulator of non-claudin-mediatedcell adhesion may be administered in conjunction with the modulatingagent(s), either within the same pharmaceutical composition orseparately. Preferred antibody modulating agents that may be used inconjunction with the claudin modulating agents include Fab fragmentsdirected against either an N-cadherin CAR sequence, such asFHLRAHAVDINGNQV-NH₂ (SEQ ID NO:25), an E-cadherin CAR sequence, such asLFSHAVSSNG-NH₂ (SEQ ID NO:26), an occludin CAR sequence, such asQYLYHYCVVD-NH₂ (SEQ ID NO:478), or an OB-cadherin CAR sequence, such asIFVIDDKSG-NH₂ (SEQ ID NO:479).

Treatment with a modulating agent may be appropriate, for example, priorto administration of an anti-tumor therapeutic or diagnostic agent(e.g., a monoclonal antibody or other macromolecule), an antimicrobialagent or an anti-inflammatory agent, in order to increase theconcentration of such agents in the vicinity of the target tumor,organism or inflammation without increasing the overall dose to thepatient. Modulating agents for use within such methods may be linked toa targeting agent to further increase the local concentration ofmodulating agent, although systemic administration of a vasoactive agenteven in the absence of a targeting agent increases the perfusion ofcertain tumors relative to other tissues. Suitable targeting agentsinclude antibodies and other molecules that specifically bind to tumorcells or to components of structurally abnormal blood vessels. Forexample, a targeting agent may be an antibody that binds to a fibrindegradation product or a cell enzyme such as a peroxidase that isreleased by granulocytes or other cells in necroticlor inflamed tissues.

Administration via intravenous injection or transdermal administrationis generally preferred. Effective dosages are generally sufficient toincrease localization of a subsequently administered diagnostic ortherapeutic agent to an extent that improves the clinical efficacy oftherapy of accuracy of diagnosis to a statistically significant degree.Comparison may be made between treated and untreated tumor host animalsto whom equivalent doses of the diagnostic or therapeutic agent areadministered. In general, dosages range as described above.

Within certain aspects, methods are provided in which cell adhesion isdiminished. In one such aspect, the present invention provides methodsfor reducing unwanted cellular adhesion in a mammal by administering amodulating agent as described herein. Unwanted cellular adhesion canoccur, for example, between tumor cells, between tumor cells and normalcells or between normal cells as a result of surgery, injury,chemotherapy, disease, inflammation or other condition jeopardizing cellviability or function. Certain preferred modulating agents for usewithin such methods comprise one or more of the claudin CAR sequencesprovided herein. In one particularly preferred embodiment, a modulatingagent is further capable of disrupting cell adhesion mediated bymultiple adhesion molecules. Such an agent may comprise, in addition toone or more claudin CAR sequences. CAR sequences such as the classicalcadherin CAR sequence HAV sequence, an RGD sequence, which is bound byintegrins; and/or the occludin CAR sequence LYHY (SEQ ID NO:17),preferably separated from the claudin CAR sequence via a linker.Alternatively, separate modulators of cell adhesion mediated by otheradhesion molecules may be administered in conjunction with themodulating agent(s), either within the same pharmaceutical compositionor separately.

Topical administration of the modulating agent(s) is generallypreferred, but other means may also be employed. Preferably, a fluidcomposition for topical administration (comprising, for example,physiological saline) comprises an amount of modulating agent asdescribed above, and more preferably from 10 μg/mL to 1 mg/mL. Creamsmay generally be formulated as described above. Topical administrationin the surgical field may be given once at the end of surgery byirrigation of the wound or as an intermittent or continuous irrigationwith the use of surgical drains in the post-operative period or by theuse of drains specifically inserted in an area of inflammation, injuryor disease in cases where surgery does not need to be performed.Alternatively, parenteral or transcutaneous administration may be usedto achieve similar results.

Within another such aspect, methods are provided for enhancing thedelivery of a drug through the skin of a mammal. Transdermal delivery ofdrugs is a convenient and non-invasive method that can be used tomaintain relatively constant blood levels of a drug. In general, tofacilitate drug delivery via the skin, it is necessary to perturbadhesion between the epithelial cells (keratinocytes) and theendothelial cells of the microvasculature. Using currently availabletechniques, only small, uncharged molecules may be delivered across skinin vivo. The methods described herein are not subject to the same degreeof limitation. Accordingly, a wide variety of drugs may be transportedacross the epithelial and endothelial cell layers of skin, for systemicor topical administration. Such drugs may be delivered to melanomas ormay enter the blood stream of the mammal for delivery to other siteswithin the body.

To enhance the delivery of a drug through the skin, a modulating agentas described herein and a drug are contacted with the skin surface.Within certain embodiments, multifunctional modulating agents comprisinga claudin CAR sequence linked to one or more of the classical cadherinCAR sequence HAV; the sequence RGD, which is bound by integrins; and/ora nonclassical cadherin CAR sequence, such as a Dsc CAR sequence (IEK,VER or IER) and/or a Dsg CAR sequence (INQ, INR or LNK), may also beused to disrupt cell adhesion. Alternatively, a separate modulator ofnon-claudin-mediated cell adhesion may be administered in conjunctionwith the modulating agent(s), either within the same pharmaceuticalcomposition or separately. Contact may be achieved by direct applicationof the modulating agent, generally within a composition formulated as acream or gel, or using any of a variety of skin contact devices fortransdermal application (such as those described in European PatentApplication No. 566,816 A; U.S. Pat. No. 5,613,958; U.S. Pat. No.5,505,956). A skin patch provides a convenient method of administration(particularly for slow-release formulations). Such patches may contain areservoir of modulating agent and drug separated from the skin by amembrane through which the drug diffuses. Within other patch designs,the modulating agent and drug may be dissolved or suspended in a polymeror adhesive matrix that is then placed in direct contact with thepatient's skin. The modulating agent and drug may then diffuse from thematrix into the skin. Modulating agent(s) and drug(s) may be containedwithin the same composition or skin patch, or may be separatelyadministered, although administration at the same time and site ispreferred. In general, the amount of modulating agent administered viathe skin varies with the nature of the condition to be treated orprevented, but may vary as described above. Such levels may be achievedby appropriate adjustments to the device used, or by applying a creamformulated as described above. Transfer of the drug across the skin andto the target tissue may be predicted based on in vitro studies using,for example, a Franz cell apparatus, and evaluated in vivo byappropriate means that will be apparent to those of ordinary skill inthe art. As an example, monitoring of the serum level of theadministered drug over time provides an easy measure of the drugtransfer across the skin.

Transdermal drug delivery as described herein is particularly useful insituations in which a constant rate of drug delivery is desired, toavoid fluctuating blood levels of a drug. For example, morphine is ananalgesic commonly used immediately following surgery. When givenintermittently in a parenteral form (intramuscular, intravenous), thepatient usually feels sleepy during the first hour, is well during thenext 2 hours and is in pain during the last hour because the blood levelgoes up quickly o after the injection and goes down below the desirablelevel before the 4 hour interval prescribed for re-injection is reached.Transdermal administrations as described herein permits the maintenanceof constant levels for long periods of time (e.g., days), which allowsadequate pain control and mental alertness at the same time. Insulinprovides another such example. Many diabetic patients need to maintainsa constant baseline level of insulin which is different from their needsat the time of meals. The baseline level may be maintained usingtransdermal administration of insulin, as described herein. Antibioticsmay also be administered at a constant rate, maintaining adequatebactericidal blood levels, while avoiding the high levels that are oftenresponsible for the toxicity (e.g., levels of gentamycin that are toohigh typically result in renal toxicity).

Drug delivery by the methods of the present invention also provide amore convenient method of drug administration. For example, it is oftenparticularly difficult to administer parenteral drugs to newborns andinfants because of the difficulty associated with finding veins ofacceptable caliber to catheterize. However, newborns and infants oftenhave a relatively large skin surface as compared to adults. Transdermaldrug delivery permits easier management of such patients and allowscertain types of care that can presently be given only in hospitals tobe given at home. Other patients who typically have similar difficultieswith venous catheterization are patients undergoing chemotherapy orpatients on dialysis. In addition, for patients undergoing prolongedtherapy, transdermal administration as described herein is moreconvenient than parenteral administration.

Transdermal administration as described herein also allows thegastrointestinal tract to be bypassed in situations where parenteraluses would not be practical. For example, there is a growing need formethods suitable for administration of therapeutic small peptides andproteins, which are typically digested within the gastrointestinaltract. The methods described herein permit administration of suchcompounds and allow easy administration over long periods of time.Patients who have problems with absorption through theirgastrointestinal tract because of prolonged ileus or specificgastrointestinal diseases limiting drug absorption may also benefit fromdrugs formulated for transdermal application as described herein.

Further, there are many clinical situations where it is difficult tomaintain compliance. For example, patients with mental problems (e.g.,patients with Alzheimer's disease or psychosis) are easier to manage ifa constant delivery rate of drug is provided without having to rely ontheir ability to take their medication at specific times of the day.Also patients who simply forget to take their drugs as prescribed areless likely to do so if they merely have to put on a skin patchperiodically (e.g., every 3 days). Patients with diseases that arewithout symptoms, like patients with hypertension, are especially atrisk of forgetting to take their medication as prescribed.

For patients taking multiple drugs, devices for transdermal applicationsuch as skin patches may be formulated with combinations of drugs thatare frequently used together. For example, many heart failure patientsare given digoxin in combination with furosemide. The combination ofboth drugs into a single skin patch facilitates administration, reducesthe risk of errors (taking the correct pills at the appropriate time isoften confusing to older people), reduces the psychological strain oftaking “so many pills,” reduces skipped dosage because of irregularactivities and improves compliance.

The methods described herein are particularly applicable to humans, butalso have a variety of veterinary uses, such as the administration ofgrowth factors or hormones (e.g., for fertility control) to an animal.

As noted above, a wide variety of drugs may be administered according tothe methods provided herein. Some examples of drug categories that maybe administered transdermally include anti-inflammatory drugs (e.g., inarthritis and in other condition) such as all NSAID, indomethacin,prednisone, etc.; analgesics (especially when oral absorption is notpossible, such as after surgery, and when parenteral administration isnot convenient or desirable), including, morphine, codeine, Demerol,acetaminophen and combinations of these (e.g., codeine plusacetaminophen); antibiotics such as Vancomycin (which is not absorbed bythe GI tract and is frequently given intravenously) or a combination ofINH and Rifampicin (e.g., for tuberculosis); anticoagulants such asheparin (which is not well absorbed by the GI tract and is generallygiven parenterally, resulting in fluctuation in the blood levels with anincreased risk of bleeding at high levels and risks of inefficacy atlower levels) and Warfarin (which is absorbed by the GI tract but cannotbe administered immediately after abdominal surgery because of thenormal ileus following the procedure); antidepressants (e.g., insituations where compliance is an issue as in Alzheimer's disease orwhen maintaining stable blood levels results in a significant reductionof anti-cholinergic side effects and better tolerance by patients), suchas amitriptylin, imipramin, prozac, etc.; antihypertensive drugs (e.g.,to improve compliance and reduce side effects associated withfluctuating blood levels), such as diuretics and beta-blockers (whichcan be administered by the same patch; e.g., furosemide and propanolol);antipsychotics (e.g., to facilitate compliance and make it easier forcare giver and family members to make sure that the drug is received),such as haloperidol and chlorpromazine; and anxiolytics or sedatives(e.g., to avoid the reduction of alertness related to high blood levelsafter oral administration and allow a continual benefit throughout theday by maintaining therapeutic levels constant).

Numerous other drugs may be administered as described herein, includingnaturally occurring and synthetic hormones, growth factors, proteins andpeptides. For example, insulin and human growth hormone, growth factorslike erythropoietin, interleukins and inteferons may be delivered viathe skin.

Kits for administering a drug via the skin of a mammal are also providedwithin the present invention. Such kits generally comprise a device fortransdermal application (e.g., a skin patch) in combination with, orimpregnated with, one or more modulating agents. A drug may additionallybe included within such kits.

Within a related aspect, the use of modulating agents as describedherein to increase skin permeability may also facilitate sampling of theblood compartment by passive diffusion, permitting detection and/ormeasurement of the levels of specific molecules circulating in theblood. For example, application of one or more modulating agents to theskin, via a skin patch as described herein, permits the patch tofunction like a sponge to accumulate a small quantity of fluidcontaining a representative sample of the serum. The patch is thenremoved after a specified amount of time and analyzed by suitabletechniques for the compound of interest (e.g., a medication, hormone,growth factor, metabolite or marker). Alternatively, a patch may beimpregnated with reagents to permit a color change if a specificsubstance (e.g., an enzyme) is detected. Substances that can be detectedin this manner include, but are not limited to, illegal drugs such ascocaine, HIV enzymes, glucose and PSA. This technology is of particularbenefit for home testing kits.

Within a further aspect, methods are provided for enhancing delivery ofa drug to a tumor in a mammal, comprising administering a modulatingagent in combination with a drug to a tumor-bearing mammal. In oneparticularly preferred embodiment, a modulating agent is capable ofdisrupting cell adhesion mediated by multiple adhesion molecules. Forexample, a single branched modulating agent (or multiple agents linkedto a single molecule or support material) may disrupt claudin, occludin,cadherin (classical and/or nonclassical, such as Dsc, Dsg, OB-cadherinand/or VE-cadherin) and integrin mediated cell adhesion, therebydisrupting tight junctions, adherens junctions, and desmosomes.Multifunctional modulating agents comprising the claudin CAR sequenceIYSY (SEQ ID NO:2) linked to one or more of the classical cadherin CARsequence HAV; the sequence RGD, which is bound, by integrins; and/or anonclassical cadherin CAR sequence, such as a Dsc CAR sequence (IEK, VERor IER), a Dsg CAR sequence (INQ, INR or LNK), an occludin CAR sequence(LYHY; SEQ ID NO:17), an OB-cadherin CAR sequence (DDK, EEY or EAQ)and/or the VE-cadherin CAR sequence DAE, may be used to disrupt celladhesion. Alternatively, a separate modulator of non-claudin-mediatedcell adhesion may be administered in conjunction with the modulatingagent(s), either within the same pharmaceutical composition orseparately. Preferred antibody modulating agents that may be used inconjunction with the claudin modulating agents include Fab fragmentsdirected against either an N-cadherin CAR sequence (such asFHLRAHAVDINGNQV-NH₂; SEQ ID NO:25) or an E-cadherin CAR sequenceLFSHAVSSNG-NH₂ (SEQ ID NO:26), an occludin CAR sequence, such asQYLYHYCVVD-NH₂ (SEQ ID NO:478), or an OB-cadherin CAR sequence, such asIFVIDDKSG-NH₂ (SEQ ID NO:479).

Preferably, the modulating agent and the drug are formulated within thesame composition or drug delivery device prior to administration. Ingeneral, a modulating agent may enhance drug delivery to any tumor, andthe method of administration may be chosen based on the type of targettumor. For example, injection or topical administration as describedabove may be preferred for melanomas and other accessible tumors (e.g.,metastases from primary ovarian tumors may be treated by flushing theperitoneal cavity with the composition). Other tumors (e.g., bladdertumors) may be treated by injection of the modulating agent and the drug(such as mitomycin C) into the site of the tumor. In other instances,the composition may be administered systemically, and targeted to thetumor using any of a variety of specific targeting agents. Suitabledrugs may be identified by those of ordinary skill in the art based uponthe type of cancer to be treated (e.g., mitomycin C for bladder cancer).In general, the amount of modulating agent administered varies with themethod of administration and the nature of the tumor, within the typicalranges provided above, preferably ranging from about 1 μg/mL to about 2mg/mL, and more preferably from about 10 μg/mL to 1 mg/mL. Transfer ofthe drug to the target tumor may be evaluated by appropriate means thatwill be apparent to those of ordinary skill in the art. Drugs may alsobe labeled (e.g., using radionuclides) to permit direct observation oftransfer to the target tumor using standard imaging techniques.

Within a related aspect, the present invention provides methods fortreating cancer and/or inhibiting metastasis in a mammal. Cancer tumorsare solid masses of cells, growing out of control, which requirenourishment via blood vessels. The formation of new capillaries is aprerequisite for tumor growth and the emergence of metastases.Administration of modulating agents as described herein may disrupt thegrowth of such blood vessels, thereby providing effective therapy forthe cancer and/or inhibiting metastasis. Modulating agents may also beused to treat leukemias. In one particularly preferred embodiment, amodulating agent is capable of disrupting cell adhesion mediated bymultiple adhesion molecules. For example, a single branched modulatingagent (or multiple agents linked to a single molecule or supportmaterial) may disrupt claudin, cadherin and integriti mediated celladhesion, thereby disrupting tight junctions, adherens junctions, focalcontacts and desmosomes. Multifunctional modulating agents comprising aclaudin CAR sequence linked to one or more of the classical cadherin CARsequence HAV; the sequence RGD, which is bound by integrins; and/or anonclassical cadherin CAR sequence, such as a Dsc CAR sequence (IEK, VERand IER), a Dsg CAR sequence (INQ, INR and/or LNK), an OB-cadherin CARsequence (DDK, EEY or EAQ), an occludin CAR sequence (LYHY; SEQ IDNO:17), and/or the VE-cadherin CAR sequence DAE, may be used to disruptcell adhesion. Alternatively, a separate modulator ofnon-claudin-mediated cell adhesion may be administered in conjunctionwith the modulating agent(s), either within the same pharmaceuticalcomposition or separately. Preferred antibody modulating agents that maybe used in conjunction with the claudin modulating agents include Fabfragments directed against either an N-cadherin CAR sequence, such asFHLRAHAVDINGNQV-NH₂ (SEQ ID NO:25), an E-cadherin CAR sequence, such asLFSHAVSSNG-NH₂ (SEQ ID NO:26), an occludin CAR sequence, such asQYLYHYCVVD-NH₂ (SEQ ID NO:478), or an OB-cadherin CAR sequence, such asIFVIDDKSG-NH₂ (SEQ ID NO:479).

A modulating agent may be administered alone (e.g., via the skin) orwithin a pharmaceutical composition. For melanomas and certain otheraccessible tumors, injection or topical administration as describedabove may be preferred. For ovarian cancers, flushing the peritonealcavity with a composition comprising one or more modulating agents mayprevent metastasis of ovarian tumor cells. Other tumors (e.g., bladdertumors, bronchial tumors or tracheal tumors) may be treated by injectionof the modulating agent into the cavity. In other instances, thecomposition may be administered systemically, and targeted to the tumorusing any of a variety of specific targeting agents, as described above.In general, the amount of modulating agent administered varies dependingupon the method of administration and the nature of the cancer, but mayvary within the ranges identified above. The effectiveness of the cancertreatment or inhibition of metastasis may be evaluated using well knownclinical observations, such as monitoring the level of serum tumormarkers (e.g., CEA or PSA).

Within a further related aspect, a modulating agent may be used toinhibit angiogenesis (i.e., the growth of blood vessels frompre-existing blood vessels) in a mammal. Inhibition of angiogenesis maybe beneficial, for example, in patients afflicted with diseases such ascancer or arthritis. In one particularly preferred embodiment, amodulating agent is capable of disrupting cell adhesion mediated bymultiple adhesion molecules. For example, a single branched modulatingagent (or multiple agents linked to a single molecule or supportmaterial) may disrupt claudin, occludin, classical cadherin, andintegrin mediated cell adhesion, thereby disrupting tight junctions,adherens junctions, and focal contacts. Multifunctional modulatingagents comprising a claudin CAR sequence linked to one or more of theclassical cadherin CAR sequence HAV, the sequence RGD, which is bound byintegrins, the OB-cadherin CAR sequence (DDK, EEY or EAQ), the occludinCAR sequence (LYHY; SEQ ID NO:17) and/or the VE-cadherin CAR sequenceDAE may be used to disrupt cell adhesion. Alternatively, a separatemodulator of non-claudin-mediated cell adhesion may be administered inconjunction with the modulating agent(s), either within the samepharmaceutical composition or separately. Preferred antibody modulatingagents that may be used in conjunction with the claudin modulatingagents include Fab fragments directed against an N-cadherin CARsequence, such as FHLRAHAVDINGNQV-NH₂ (SEQ ID NO:25), an occludin CARsequence, such as QYLYHYCVVD-NH₂ (SEQ ID NO:478), or an OB-cadherIFVIDDKSG-NH₂ (SEQ ID NO:479).

The effect of a particular modulating agent on angiogenesis maygenerally be determined by evaluating the effect of the agent on bloodvessel formation. Such a determination may generally be performed, forexample, using a chick chorioallantoic membrane assay (Iruela-Arispe etal., Molecular Biology of the Cell 6:327-343, 1995). Briefly, amodulating agent may be embedded in a mesh composed of vitrogen at oneor more concentrations (e.g., ranging from about 5 to 50 μg/mesh). Themesh(es) may then be applied to chick chorioallantoic membranes. After24 hours, the effect of the modulating agent may be determined usingcomputer assisted morphometric analysis. A modulating agent shouldinhibit angiogenesis by at least 25% at a concentration of 50 μg/mesh.

The addition of a targeting agent as described above may be beneficial,particularly when the administration is systemic. Suitable modes ofadministration and dosages depend upon the condition to be prevented ortreated but, in general, administration by injection is appropriate.Dosages may vary as described above. The effectiveness of the inhibitionmay be evaluated grossly by assessing the inability of the tumors tomaintain their growth and microscopically by observing an absence ofnerves at the periphery of the tumor.

In yet another related aspect, the present invention provides methodsfor inducing apoptosis in a claudin-expressing cell. In general,patients afflicted with cancer may benefit from such treatment. In oneparticularly preferred embodiment, a modulating agent is capable ofdisrupting cell adhesion mediated by multiple adhesion molecules. Forexample, a single branched modulating agent (or multiple agents linkedto a single molecule or support material) may disrupt claudin occludin,classical cadherin, and integrin mediated cell adhesion, therebydisrupting tight junctions, adherens junctions, and focal contacts.Multifunctional modulating agents comprising a claudin CAR sequencelinked to one or more of the cadherin CAR sequence HAV and/or thesequence RGD, which is bound by integrins, and occludin CAR sequenceLYHY (SEQ ID NO:17) may be used to disrupt cell adhesion. Alternatively,a separate modulator of non-claudin-mediated cell adhesion may beadministered in conjunction with the modulating agent(s), either withinthe same pharmaceutical composition or separately. Preferred antibodyimodulating agents that may be used in conjunction with the claudinmodulating agents include Fab fragments directed against either anN-cadherin CAR sequence, such as FHLRAHAVDINGNQV-NH₂ (SEQ ID NO:25), oran E-cadherin CAR sequence, such as LFSHAVSSNG-NH₂ (SEQ ID NO:26), anoccludin CAR sequence, such as QYLYHYCVVD-NH₂ (SEQ ID NO:478),: or anOB-cadherin CAR sequence, such as IFVIDDKSG-NH₂ (SEQ ID NO:479).

Administration of modulating agents to induce apoptosis may be topical,via injection or by other means and the addition of a targeting agentmay be beneficial, particularly when the administration is systemic.Suitable modes of administration and dosages depend upon the locationand nature of the cells for which induction of apoptosis is desired but,in general, dosages may vary as described above. A biopsy may beperformed to evaluate the level of induction of apoptosis.

The present invention also provides methods for enhancing drug deliveryto the central nervous system of a mammal. The blood/brain barrier islargely impermeable to most neuroactive agents, and delivery of drugs tothe brain of a mammal often requires invasive procedures. Using amodulating agent as described herein, however, delivery may be by, forexample, systemic administration of a modulating agent-drug-targetingagent combination, injection of a modulating agent (alone or incombination with a drug and/or targeting agent) into the carotid arteryor application of a skin patch comprising a modulating agent to the headof the patient. In one particularly preferred embodiment, a modulatingagent is capable of disrupting cell adhesion mediated by multipleadhesion molecules. For example, a single branched modulating agent (ormultiple agents linked to a single molecule or support material) maydisrupt claudin, occludin and cadherin mediated cell adhesion, therebydisrupting tight junctions and adherens junctions. Multi-functionalmodulating agents comprising a claudin CAR sequence linked to one ormore of the classical cadherin CAR sequence HAV, the occludin CARsequence LYHY (SEQ ID NO:17), the OB-cadherin CAR sequence (DDK, EEY orEAQ) and/or the VE-cadherin CAR sequence DAE, preferably by way of alinker, are also preferred. Alternatively, a separate modulator ofnon-claudin-mediated cell adhesion may be administered in conjunctionwith the modulating agent(s), either within the same pharmaceuticalcomposition or separately. Preferred antibody modulating agents that maybe used in conjunction with the claudin modulating agents include Fabfragments directed against the N-cadherin CAR sequenceFHLRAHAVDINGNQV-NH₂ (SEQ ID NO:25), an occludin CAR sequence, such asQYLYHYCVVD-NR₂ (SEQ ID NO:478), or an OB-cadherin CAR sequence, such asIFVIDDKSG-NH₂ (SEQ ID NO:479).

In general, the amount of modulating agent administered varies with themethod of administration and the nature of the condition to be treatedor prevented, but typically varies as described above. Transfer of thedrug to the central nervous system may be evaluated by appropriate meansthat will be apparent to those of ordinary skill in the art, such asmagnetic resonance imaging (MRI) or PET scan (positron emittedtomography).

In certain other aspects, the present invention provides methods forenhancing adhesion of claudin-expressing cells. Within certainembodiments, a modulating agent may be linked to a solid support,resulting in a matrix that comprises multiple modulating agents. Withinone such embodiment, the support is a polymeric matrix to whichmodulating agents and molecules comprising other CAR sequence(s) areattached (e.g., modulating agents and molecules comprising HAV and RGDsequences may be attached to the same matrix, preferably in analternating pattern). Such matrices may be used in contexts in which itis desirable to enhance adhesion mediated by multiple cell adhesionmolecules. Alternatively, the modulating agent itself may comprisemultiple claudin CAR sequences or antibodies (or fragments thereof),separated by linkers as described above. Either way, the modulatingagent(s) function as a “biological glue” to bind multipleclaudin-expressing cells within a variety of contexts.

Within one such aspect, modulating agents comprising multiple claudinCAR sequences and/or multiple modulating agents linked to a singlemolecule or support material may be used to enhance wound healing and/orreduce scar tissue in a mammal. Modulating agents that are linked to abiocompatible and biodegradable matrix such as cellulose or collagen areparticularly preferred. For use within such methods, a modulating agentshould have a free amino or hydroxyl group. The modulating agents aregenerally administered topically to the wound, where they may facilitateclosure of the wound and may augment, or even replace, stitches.Similarly, administration of matrix-linked modulating agents mayfacilitate cell adhesion in skin grafting and prosthetic implants, andmay prolong the duration and usefulness of collagen injection. Ingeneral, the amount of matrix-linked modulating agent administered to awound, graft or implant site varies with the severity of the woundand/or the nature of the wound, graft, or implant, but may vary asdiscussed above. Multi-functional modulating agents comprising theclaudin CAR sequence, IYSY (SEQ ID NO:2), the classical cadherin CARsequence (HAV), the integrin CAR sequence (RGD), the occludin CARsequence LYHY (SEQ ID NO:17), as well as a nonclassical cadherin CARsequence, such as the OB-cadherin CAR sequence (DDK, EEY or EAQ), theVE-cadherin CAR sequence DAE and/or one or more of the Dsc and Dsg CARsequences IEK, VER, IER, INQ, INR and/or LNK, may also be used as potentstimulators of wound healing and/or to reduce scar tissue.Alternatively, one or more separate modulator of cadherin-, integrin-,and/or nonclassical cadherin-mediated cell adhesion may be administeredin conjunction with the modulating agent(s), either within the samepharmaceutical composition or separately.

Within another aspect, one or more modulating agents may be linked tothe interior surface of a tissue culture plate or other cell culturesupport, such as for use in a bioreactor. Such linkage may be performedby any suitable technique, as described above. Modulating agents linkedin this fashion may generally be used to immobilize claudin-expressingcells. For example, dishes or plates coated with one or more modulatingagents may be used to immobilize claudin-expressing cells within avariety of assays and screens. Within bioreactors (i.e., systems forlarge scale production of cells or organoids), modulating agents maygenerally be used to improve cell attachment and stabilize cell growth.Modulating agents may also be used within bioreactors to support theformation and function of highly differentiated organoids derived, forexample, from dispersed populations of fetal mammalian cells.Bioreactors containing biomatrices of modulating agent(s) may also beused to facilitate the production of specific proteins.

Modulating agents as described herein may be used within a variety ofbioreactor configurations. In general, a bioreactor is designed with aninterior surface area sufficient to support large numbers of adherentcells. This surface area can be provided using membranes, tubes,microtiter wells, columns, hollow fibers, roller bottles, plates,dishes, beads or a combination thereof. A bioreactor may becompartmentalized. The support material within a bioreactor may be anysuitable material known in the art; preferably, the support materialdoes not dissolve or swell in water. Preferred support materialsinclude, but are not limited to, synthetic polymers such as acrylics,vinyls, polyethylene, polypropylene, polytetrafluoroethylene, nylons,polyurethanes, polyamides, polysulfones and poly(ethyleneterephthalate); ceramics; glass and silica.

Other aspects of the present invention provide methods that employantibodies raised against the modulating agents for diagnostic and assaypurposes. Assays typically involve using an antibody to detect thepresence or absence of claudin (free or on the surface of a cell), orproteolytic fragment containing the EC1 domain in a suitable biologicalsample, such as tumor or normal tissue biopsies, blood, lymph node,serum or urine samples, or other tissue, homogenate, or extract thereofobtained from a patient.

There are a variety of assay formats known to those of ordinary skill inthe art for using an antibody to detect a target molecule in a sample.See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, 1988. For example, the assay may be performed in aWestern blot format, wherein a protein preparation from the biologicalsample is submitted to gel electrophoresis, transferred to a suitablemembrane and allowed to react with the antibody. The presence of theantibody on the membrane may then be detected using a suitable detectionreagent, as described below.

In another embodiment, the assay involves the use of antibodyimmobilized on a solid support to bind to the target claudin, or aproteolytic fragment containing the EC1 domain and encompassing the CARsequence, and remove it from the remainder of the sample. The boundclaudin may then be detected using a second antibody or reagent thatcontains a reporter group. Alternatively, a competitive assay may beutilized, in which the claudin is labeled with a reporter group andallowed to bind to the immobilized antibody after incubation of theantibody with the sample. The to extent to which components of thesample inhibit the binding of the labeled claudin to the antibody isindicative of the reactivity of the sample with the immobilizedantibody, and as a result, indicative of the level of the claudin in thesample.

The solid support may be any material known to those of ordinary skillin the art to which the antibody may be attached, such as a test well ina microtiter plate, a nitrocellulose filter or another suitablemembrane. Alternatively, the support may be a bead or disc, such asglass, fiberglass, latex or a plastic such as polystyrene orpolyvinylchloride. The antibody may be immobilized on the solid supportusing a variety of techniques known to those in the art, which are amplydescribed in the patent and scientific literature.

In certain embodiments, the assay for detection of claudin in a sampleis a two-antibody sandwich assay. This assay may be performed by firstcontacting an antibody that has been immobilized on a solid support,commonly the well of a microtiter plate, with the biological sample,such that the claudin within the sample is allowed to bind to theimmobilized antibody (a 30 minute incubation time at room temperature isgenerally sufficient). Unbound sample is then removed from theimmobilized claudin-antibody complexes and a second antibody(;containing a reporter group such as an enzyme, dye, radionuclide,luminescent group, fluorescent group or biotin) capable of binding to adifferent site on the claudin is added. The amount of second antibodythat remains bound to the solid support is then determined using amethod appropriate for the specific reporter group. The method employedfor detecting the reporter group depends upon the nature of the reportergroup. For radioactive groups, scintillation counting orautoradiographic methods are generally appropriate. Spectroscopicmethods may be used to detect dyes, luminescent groups and fluorescentgroups. Biotin may be detected using avidin, coupled to a differentreporter group (commonly a radioactive or fluorescent group or anenzyme). Enzyme reporter groups may generally be detected by theaddition of substrate (generally for a specific period of time),followed by spectroscopic or other analysis of the reaction products.Standards and standard additions may be used to determine the level ofclaudin in a sample, using well known techniques.

The present invention also provides kits for use in such immunoassays.Such kits generally comprise one or more antibodies, as described above.In addition, one or more additional compartments or containers of a kitgenerally enclose elements, such as reagents, buffers and/or washsolutions, to be used in the immunoassays.

Within further aspects, modulating agents or antibodies (or fragmentsthereof) may be used to facilitate cell identification and sorting invitro or imaging in vivo, permitting the selection of cells expressingclaudin (or different claudin levels). Preferably, the modulatingagent(s) or antibodies for use in such methods are linked to adetectable marker. Suitable markers are well known in the art andinclude radionuclides, luminescent groups, fluorescent groups, enzymes,dyes, constant immunoglobulin domains and biotin. Within one preferredembodiment, a modulating agent linked to a fluorescent marker, such asfluorescein, is contacted with the cells, which are then analyzed byfluorescence activated cell sorting (FACS).

Antibodies or fragments thereof may also be used within screens ofcombinatorial or other nonpeptide-based libraries to identify othercompounds capable, of modulating claudin-mediated cell adhesion. Suchscreens may generally be performed using an ELISA or other method wellknown to those of ordinary skill in the art that detect compounds with ashape and structure similar to that of the modulating agent. In general,such screens may involve contacting an expression library producing testcompounds with an antibody, and detecting the level of antibody bound tothe candidate compounds. Compounds for which the antibody has a higheraffinity may be further characterized as described herein, to evaluatethe ability to modulate claudin-mediated cell adhesion.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLE 1 Preparation of Representative Cyclic Peptides

This Example illustrates the solid phase synthesis of representativelinear and cyclic peptides as modulating agents.

The peptides are assembled on methylbenzhydrylamine resin (MBHA resin)for the C-terminal amide peptides. The traditional Merrifield resins areused for any C-terminal acid peptides. Bags of a polypropylene meshmaterial are filled with the resin and soaked in dichloromethane. Theresin packets are washed three times with 5% diisopropylethylamine indichloromethane and then washed with dichloromethane. The packets arethen sorted and placed into a Nalgene bottle containing a solution ofthe amino acid of interest in dichloromethane. An equal amount ofdiisopropylcarbodiimide (DIC) in dichloromethane is added to activatethe coupling reaction. The bottle is shaken for one hour to ensurecompletion of the reaction. The reaction mixture is discarded and thepackets washed with DMF. The N-α-Boc is removed by acidolysis using a55% TFA in dichloromethane for 30 minutes. leaving the TFA salt of theα-amino group. The bags are washed and the synthesis completed byrepeating the same procedure while substituting for the correspondingamino acid at the coupling step. Acetylation of the N-terminal isperformed by reacting the peptide resins with a solution of aceticanhydride in dichloromethane in the presence of diisopropylethylamine.The peptide is then side-chain deprotected and cleaved from the resin at0° C. with liquid HF in the presence of anisole as a carbocationscavenger.

The crude peptides are purified by reversed-phase high-performanceliquid chromatography. Purified linear precursors of the cyclic peptidesare solubilized in 75% acetic acid at a concentration of 2-10 mg/mL. A10% solution of iodine in methanol is added dropwise until a persistentcoloration was obtained. A 5% ascorbic acid solution in water is thenadded to the mixture until discoloration. The disulfide bridgecontaining compounds are then purified by HPLC and characterized byanalytical HPLC and by mass spectral analysis.

EXAMPLE 2 System for Assessing Endothelial Cell Adhesion

This Example illustrates an endothelial cell adhesion assay forevaluating the effects of modulating agents on endothelial celladhesion.

A. Cell Culture

Human aortic endothelial cells (HAEC) are cultured on fibronectin(Sigma, St. Louis, Mo.) according to the procedures of Jaffe et al., J.Clin. Invest. 52:2745-2756, 1973. Cells are maintained in EGM(endothelial cell growth medium; Clonetics, San Diego, Calif.) and usedfor experiments at passage 4.

B. Claudin and VE-cadherin Immunolocalization Methods

HAEC are cultured on fibronectin-coated coverslips. Confluent culturesof HAEC are exposed to linear peptides (final concentration 100 μg/mlEGM), or EGM alone for 1 hour. The cells are then fixed for 30 minutesat 4° C. in 95% ethanol; followed by fixation in acetone for 1 minute at4° C. (Furuse et al., J. Cell Biol. 123:1777-1788, 1993). Afterfixation, the cells are allowed to air dry at room temperature. Thecells are probed with either mouse anti-VE-cadherin antibodies (Hemeris,Sassenage, France; diluted 1:250 in 0.1% dried skim milk powderdissolved in PBS), or anti-claudin antibodies (prepared as described byMorita et al., Proc. Natl. Acad Sci. USA 96:511-516, 1999, and generallyas described in Blaschuk and Farookhi, Dev. Biol. 136:564-567, 1989)diluted in 0.1% dried skim milk powder dissolved in PBS) for 1 hour at37° C. Briefly, a nine amino acid peptide comprising a claudin CARsequence is linked to KLH and injected into rabbits. The animals arebled, and polyclonal antibodies specific for the CAR sequence arepurified from the antisera by affinity chromatography using the peptidecoupled to a solid support.

The cells are then washed with 0.1% dried skim milk powder dissolved inPBS (three washes, 5 minutes/wash), and probed with secondary antibodies(donkey anti-mouse Cy3, or donkey anti-rabbit Cy5 diluted 1:250 in 0.1%dried skim milk powder dissolved in PBS; Jackson ImmunoresearchLaboratories Inc., Westgrove, Pa.) for 1 hour at 37° C. The cells arewashed again with in 0.1% dried skim milk powder dissolved in PBS andmounted in a solution composed of 50% glycerol and 50% PBS to whichphenylenediamine (Sigma, St. Louis, Mo.) had been added to a finalconcentration of 1 mg/ml. The samples are analyzed using a Bio-Rad MRC1000 confocal microscope with Laser Sharp software version 2.1T(Bio-Rad, Hercules, Calif.). Staining for claudin is assigned thepseudo-color red, whereas VE-cadherin staining is assigned pseudo-colorgreen using Confocal Assistant 4.02 software. The endothelial cells areseen to retract from one another when cultured in the presence of amodulating agent comprising a claudin CAR sequence, indicating thatadhesion is decreased between the cells. Furthermore, the cells do notform cobblestone-like monolayers when exposed to such an agent. Surfaceexpression of both VE-cadherin and claudin is greatly reduced in thecells treated with modulating agent, as compared to the VE-cadherin andclaudin levels expressed by untreated cells.

EXAMPLE 3 Assay for Evaluating Effect of Representative ModulatingAgents on Vasopermeability

This Example illustrates a vasopermeability assay for evaluating theeffects of modulating agents on endothelial cell permeability in vivo.

A. Miles Assay for Vascular Permeability

The ability of cyclic and linear peptides to increase vascularpermeability is assessed utilizing the Miles assay (McClure et al., J.Pharmacological & Toxicological Meth. 32:49-521994). The peptides aredissolved in phosphate buffered saline (PBS) at a concentration of 100μg/ml. Adult rats are given 100 μl subdermal injections of each peptidesolution into their shaved backs, followed 15 minutes later by a single250 μl injection of 1% Evans blue dissolved in PBS into their tailveins. The subdermal injection sites are visually monitored for theappearance of blue dye. Once the dye appears, each subdermal injectionsite is excised, weighed, and placed 1 ml dimethylformamide for 24 hoursto extract the dye. The optical density of the dye extracts isdetermined at 620 nm. More blue dye is seen to accumulate at sites wherethe peptide was injected, as opposed to sites where phosphate bufferedsaline was injected.

EXAMPLE 4 Assay for Evaluating Effect of Representative ModulatingAgents on NRK Cell Adhesion

This Example illustrates an assay for evaluating the effects ofmodulating agents on adhesion of NRK cells.

NRK cells (ATCC #1571-CRL) are plated at 10-20,000 cells per 35 mmtissue culture flasks containing DMEM with 10% FCS and sub-culturedperiodically (Laird et al., J. Cell Biol. 131:1193-1203, 1995). Cellsare harvested and replated in 35 mm tissue culture flasks containing 1mm coverslips and incubated until 50-65% confluent (24-36 hours). Atthis time, coverslips are transferred to a 24-well plate, washed oncewith fresh DMEM and exposed to modulating agent at a concentration of0.1 mg/mL for 24 hours. Fresh modulating agent is then be added, and thecells left for an additional 24 hours. Cells are fixed with 100%methanol for 10 minutes and then washed three times with PBS. Coverslipsare blocked for 1 hour in 2% BSA/PBS and incubated for a further 1 hourin the presence of anti-claudin antibody, as described above, and mouseanti-E-cadherin antibody (Transduction Labs, 1:250 dilution). Primaryand secondary antibodies are diluted in 2% BSA/PBS. Following incubationin the primary antibody, coverslips are washed three times for 5 minuteseach in PBS and incubated for 1 hour with donkey anti-mouse Cy3 anddonkey anti-rabbit Cy5 (Jackson Immunoresearch Laboratories Inc.,Westgrove, Pa.) for 1 hour at 37° C. Following further washes in PBS(3×5 min) coverslips are mounted and viewed by confocal microscopy.

In the absence of modulating agent, NRK cells form characteristictightly adherent monolayers with a cobblestone morphology in which cellsdisplay a polygonal shape. NRK cells that are treated with a modulatingagent that disrupts claudin-mediated cell adhesion assume anon-polygonal and elongated morphology (i.e., a fibroblast-like shape)within 48 hours of treatment with 0.1 mg/mL of modulating agent. Gapsappear in confluent cultures of such cells. In addition, 0.1 mg/mL ofsuch a modulating agent reproducibly induces a readily apparentreduction in cell surface staining of claudin and E-cadherin, as judgedby immunofluorescence microscopy (Laird et al., J. Cell Biol.131:1193-1203, 1995) within 48 hours.

EXAMPLE 5 Effect of Representative Modulating Agents on ElectricalResistance Across Cell Monolayer

This Example illustrates an electrical resistance assay for evaluatingthe effects of claudin-modulating agents on epithelial cell adhesion.

Madin Darby canine kidney (MDCK) cells were plated in Millicells(Millipore, Bedford, Mass.), at a density of 300,000 cells perMillicell, and cultured in Dulbecco's Modified Eagle Medium (DMEM;Sigma, St. Louis, Mo.) containing 5% fetal calf serum (Sigma, St. Louis,Mo.) until monolayers formed. Monolayers were exposed to the modulatingagent dissolved in medium. The electrical resistance was measured usingthe EVOM device (World Precision Instruments, Sarasota, Fla.). At thetime of measurement, fresh medium, with or without the modulating agent,may be added to the Millicells.

FIG. 3 shows the mean electrical resistance across MDCK cell monolayerscultured for 18 hours in medium alone (Control), medium containingN—Ac-WKIYSYAGDN-NH₂ (Peptide 118, SEQ ID NO:475) or H-WKIYSYAGDN-NH₂(Peptide 119; SEQ ID NO:475) at a concentration of 0.5 mg/ml. Duplicatemeasurements were taken, and error bars represent the standarddeviation. Peptide 118 reduced the electrical resistance across themonolayer, while peptide 119 did not change the electrical resistanceacross the monolayer relative to the control.

FIG. 4 shows the mean electrical resistance across MDCK cell monolayerscultured for 24 hours in medium alone (Control) or medium containingN—Ac-WKIYSYAGDN-NH₂ (Peptide 118; SEQ ID NO:475) at variousconcentrations. Peptide 118 reduced the electrical resistance across themonolayer in a dose dependent manner.

These results demonstrate the ability of modulating agents to inhibitthe formation of tight junctions in epithelial cells, as well as theeffect of the N—Ac group of activity of this particular modulatingagent.

From the foregoing, it will be evident that although specificembodiments of the invention have been described herein for the purposeof illustrating the invention, various modifications may be made withoutdeviating from the spirit and scope of the invention. Accordingly, theinvention is not limited except as by the appended claims.

531 1 8 PRT Unknown MOD_RES (2) Where Xaa is either Lysine, Arginine orGlutamine 1 Trp Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 2 4 PRT ArtificialSequence Description of Artificial Sequence Product of Synthesis basedon Mouse Claudin-1 Cell Adhesion Recognition Sequence 2 Ile Tyr Ser Tyr1 3 4 PRT Artificial Sequence Description of Artificial Sequence Productof synthesis based on mouse claudin-2 cell adhesion recognition sequence3 Thr Ser Ser Tyr 1 4 4 PRT Mus musculus Description of ArtificialSequence Product of synthesis based on human, mouse and Monkey CPE-Rcell adhesion recognition sequence 4 Val Thr Ala Phe 1 5 4 PRTArtificial Sequence Description of Artificial Sequence Product ofsynthesis based on human and rat RVP-1 cell adhesion recognitionsequence 5 Val Ser Ala Phe 1 6 42 PRT Mus musculus 6 Pro Gln Trp Lys IleTyr Ser Tyr Ala Gly Asp Asn Ile Val Thr Ala 1 5 10 15 Gln Ala Ile TyrGlu Gly Leu Trp Met Ser Cys Val Ser Gln Ser Thr 20 25 30 Gly Gln Ile GlnCys Lys Val Phe Asp Ser 35 40 7 42 PRT Mus musculus 7 Pro Asn Trp ArgThr Ser Ser Tyr Val Gly Ala Ser Ile Val Thr Ala 1 5 10 15 Val Gly PheSer Lys Gly Leu Trp Met Glu Cys Ala Thr His Ser Thr 20 25 30 Gly Ile ThrGln Cys Asp Ile Tyr Ser Thr 35 40 8 42 PRT Homo sapiens 8 Pro Met TrpArg Val Thr Ala Phe Ile Gly Ser Asn Ile Val Thr Ser 1 5 10 15 Gln ThrIle Trp Glu Gly Leu Trp Met Asn Cys Val Val Gln Ser Thr 20 25 30 Gly GlnMet Gln Cys Lys Val Tyr Asp Ser 35 40 9 42 PRT Mus musculus 9 Pro MetTrp Arg Val Thr Ala Phe Ile Gly Ser Asn Ile Val Thr Ala 1 5 10 15 GlnThr Ser Trp Glu Gly Leu Trp Met Asn Cys Val Val Gln Ser Thr 20 25 30 GlyGln Met Gln Cys Lys Met Tyr Asp Ser 35 40 10 42 PRT Chlorocebus aethiops10 Pro Met Trp Arg Val Thr Ala Phe Ile Gly Ser Asn Ile Val Thr Ser 1 510 15 Gln Thr Ile Trp Glu Gly Leu Trp Met Asn Cys Val Val Gln Ser Thr 2025 30 Gly Gln Met Gln Cys Lys Val Tyr Asp Ser 35 40 11 42 PRT Homosapiens 11 Pro Met Trp Arg Val Ser Ala Phe Ile Gly Ser Asn Ile Ile ThrSer 1 5 10 15 Gln Asn Ile Trp Glu Gly Leu Trp Met Asn Cys Val Val GlnSer Thr 20 25 30 Gly Gln Met Gln Cys Lys Val Tyr Asp Ser 35 40 12 41 PRTRattus norvegicus 12 Pro Met Trp Arg Val Ser Ala Phe Ile Gly Ser Ser IleIle Thr Ala 1 5 10 15 Gln Ile Thr Trp Glu Gly Leu Trp Met Asn Cys ValGln Ser Thr Gly 20 25 30 Gln Met Gln Cys Lys Met Tyr Asp Ser 35 40 13 42PRT Unknown Description of Unknown Organism Consensus Claudinextracellular domain 1 sequence 13 Pro Xaa Trp Xaa Xaa Xaa Xaa Xaa XaaGly Xaa Xaa Ile Xaa Thr Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Gly Leu TrpMet Xaa Cys Xaa Xaa Xaa Xaa Thr 20 25 30 Gly Xaa Xaa Gln Cys Xaa Xaa XaaXaa Xaa 35 40 14 8 PRT Artificial Sequence Description of ArtificialSequence Product of Synthesis based on mousle claudin-1 sequence 14 IleTyr Ser Tyr Ile Tyr Ser Tyr 1 5 15 15 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on mouseclaudin-1 sequence 15 Gln Ile Tyr Ser Tyr Gln Ile Tyr Ser Tyr Gln IleTyr Ser Tyr 1 5 10 15 16 10 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis. N-CAM binding sequence 16 LysTyr Ser Phe Asn Tyr Asp Gly Ser Glu 1 5 10 17 4 PRT Artificial SequenceDescription of Artificial Sequence Product of Syntheis. Occludin celladhesion recognition sequence 17 Leu Tyr His Tyr 1 18 6 PRT ArtificialSequence Description of Artificial Sequence Product of synthesis basedon mouse claudin-1 sequence 18 Cys Ile Tyr Ser Tyr Xaa 1 5 19 6 PRTArtificial Sequence Cyclic Peptide 19 Xaa Ile Tyr Ser Tyr Cys 1 5 20 6PRT Artificial Sequence Description of Artificial Sequence Product ofSynthesis based on mouse claudin-1 sequence 20 Xaa Ile Tyr Ser Tyr Cys 15 21 6 PRT Artificial Sequence Description of Artificial SequenceProduct of synthesis based on mouse claudin-1 sequence 21 Xaa Ile TyrSer Tyr Cys 1 5 22 6 PRT Artificial Sequence Description of ArtificialSequence Product of Synthesis based on mouse claudin-1 sequence 22 XaaIle Tyr Ser Tyr Cys 1 5 23 5 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on mouse claudin-1sequence 23 Xaa Ile Tyr Ser Tyr 1 5 24 4 PRT Artificial SequenceDescription of Artificial Sequence Product of Synthesis 24 Trp Gly GlyTrp 1 25 15 PRT Artificial Sequence Description of Artificial SequenceProduct of Synthesis based on N-cadherin cell adhesion recognitionsequence 25 Phe His Leu Arg Ala His Ala Val Asp Ile Asn Gly Asn Gln Val1 5 10 15 26 10 PRT Artificial Sequence Description of ArtificialSequence Product of synthesis based on E-cadherin cell adhesionrecognition sequence 26 Leu Phe Ser His Ala Val Ser Ser Asn Gly 1 5 1027 5 PRT Artificial Sequence Description of Artificial Sequence Productof Synthesis based on mouse claudin-1 sequence 27 Ile Tyr Ser Tyr Ala 15 28 6 PRT Artificial Sequence Description of Artificial SequenceProduct of Synthesis based on mouse claudin-1 sequence 28 Ile Tyr SerTyr Ala Gly 1 5 29 5 PRT Artificial Sequence Description of ArtificialSequence Product of Synthesis based on mouse claudin-1 sequence 29 LysIle Tyr Ser Tyr 1 5 30 6 PRT Artificial Sequence Description ofArtificial Sequence Product of Synthesis based on mouse claudin-1sequence 30 Lys Ile Tyr Ser Tyr Ala 1 5 31 7 PRT Artificial SequenceDescription of Artificial Sequence Product of Synthesis based on mouseclaudin-1 sequence 31 Lys Ile Tyr Ser Tyr Ala Gly 1 5 32 6 PRTArtificial Sequence Description of Artificial Sequence Product ofSynthesis based on mouse claudin-1 sequence 32 Trp Lys Ile Tyr Ser Tyr 15 33 7 PRT Artificial Sequence Description of Artificial SequenceProduct of Synthesis based on mouse claudin-1 sequence 33 Trp Lys IleTyr Ser Tyr Ala 1 5 34 8 PRT Artificial Sequence Description ofArtificial Sequence Product of Synthesis based on mouse claudin-1sequence 34 Trp Lys Ile Tyr Ser Tyr Ala Gly 1 5 35 5 PRT ArtificialSequence Description of Artificial Sequence Product of synthesis basedon mouse claudin-2 sequence 35 Thr Ser Ser Tyr Val 1 5 36 6 PRTArtificial Sequence Description of Artificial Sequence Product ofsynthesis based on mouse claudin-2 sequence 36 Thr Ser Ser Tyr Val Gly 15 37 5 PRT Artificial Sequence Description of Artificial SequenceProduct of synthesis based on mouse claudin-2 sequence 37 Arg Thr SerSer Tyr 1 5 38 6 PRT Artificial Sequence Description of ArtificialSequence Product of synthesis based on mouse claudin-2 sequence 38 ArgThr Ser Ser Tyr Val 1 5 39 7 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on mouse claudin-2sequence 39 Arg Thr Ser Ser Tyr Val Gly 1 5 40 6 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on mouseclaudin-2 sequence 40 Trp Arg Thr Ser Ser Tyr 1 5 41 7 PRT ArtificialSequence Description of Artificial Sequence Product of synthesis basedon mouse claudin-2 sequence 41 Trp Arg Thr Ser Ser Tyr Val 1 5 42 8 PRTArtificial Sequence Description of Artificial Sequence Product ofsynthesis based on mouse claudin-2 sequence 42 Trp Arg Thr Ser Ser TyrVal Gly 1 5 43 5 PRT Artificial Sequence Description of ArtificialSequence Product of synthesis based on human, mouse and monkey CPE-Rsequences 43 Val Thr Ala Phe Ile 1 5 44 6 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on human,mouse and monkey CPE-R sequences 44 Val Thr Ala Phe Ile Gly 1 5 45 5 PRTArtificial Sequence Description of Artificial Sequence Product ofsynthesis based on human, mouse and monkey CPE-R sequences 45 Arg ValThr Ala Phe 1 5 46 6 PRT Artificial Sequence Description of ArtificialSequence Product of synthesis based on human, mouse and monkey CPE-Rsequences 46 Arg Val Thr Ala Phe Ile 1 5 47 7 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on human,mouse and monkey CPE-R sequences 47 Arg Val Thr Ala Phe Ile Gly 1 5 48 6PRT Artificial Sequence Description of Artificial Sequence Product ofsynthesis based on human, mouse and monkey CPE-R sequences 48 Trp ArgVal Thr Ala Phe 1 5 49 7 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on human, mouse andmonkey CPE-R sequences 49 Trp Arg Val Thr Ala Phe Ile 1 5 50 8 PRTArtificial Sequence Description of Artificial Sequence Product ofsynthesis based on human, mouse and monkey CPE-R sequences 50 Trp ArgVal Thr Ala Phe Ile Gly 1 5 51 5 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on human and rat RVP-1sequences 51 Val Ser Ala Phe Ile 1 5 52 6 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on humanand rat RVP-1 sequences 52 Val Ser Ala Phe Ile Gly 1 5 53 5 PRTArtificial Sequence Description of Artificial Sequence Product ofsynthesis based on human and rat RVP-1 sequences 53 Arg Val Ser Ala Phe1 5 54 6 PRT Artificial Sequence Description of Artificial SequenceProduct of synthesis based on human and rat RVP-1 sequences 54 Arg ValSer Ala Phe Ile 1 5 55 7 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on human and rat RVP-1sequences 55 Arg Val Ser Ala Phe Ile Gly 1 5 56 6 PRT ArtificialSequence Description of Artificial Sequence Product of synthesis basedon human and rat RVP-1 sequences 56 Trp Arg Val Ser Ala Phe 1 5 57 7 PRTArtificial Sequence Description of Artificial Sequence Product ofsynthesis based on human and rat RVP-1 sequences 57 Trp Arg Val Ser AlaPhe Ile 1 5 58 8 PRT Artificial Sequence Description of ArtificialSequence Product of synthesis based on human and rat RVP-1 sequences 58Trp Arg Val Ser Ala Phe Ile Gly 1 5 59 6 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on mouseclaudin-1 sequence 59 Cys Ile Tyr Ser Tyr Cys 1 5 60 7 PRT ArtificialSequence Description of Artificial Sequence Product of synthesis basedon mouse claudin-1 sequence 60 Cys Ile Tyr Ser Tyr Ala Cys 1 5 61 8 PRTArtificial Sequence Description of Artificial Sequence Product ofsynthesis based on mouse claudin-1 sequence 61 Cys Ile Tyr Ser Tyr AlaGly Cys 1 5 62 7 PRT Artificial Sequence Description of ArtificialSequence Product of synthesis based on mouse claudin-1 sequence 62 CysLys Ile Tyr Ser Tyr Cys 1 5 63 8 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on mouse claudin-1sequence 63 Cys Lys Ile Tyr Ser Tyr Ala Cys 1 5 64 9 PRT ArtificialSequence Description of Artificial Sequence Product of synthesis basedon mouse claudin-1 sequence 64 Cys Lys Ile Tyr Ser Tyr Ala Gly Cys 1 565 8 PRT Artificial Sequence Description of Artificial Sequence Productof synthesis based on mouse claudin-1 sequence 65 Cys Trp Lys Ile TyrSer Tyr Cys 1 5 66 9 PRT Artificial Sequence Description of ArtificialSequence Product of synthesis based on mouse claudin-1 sequence 66 CysTrp Lys Ile Tyr Ser Tyr Ala Cys 1 5 67 10 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on mouseclaudin-1 sequence 67 Cys Trp Lys Ile Tyr Ser Tyr Ala Gly Cys 1 5 10 686 PRT Artificial Sequence Description of Artificial Sequence Product ofsynthesis based on mouse claudin-1 sequence 68 Lys Ile Tyr Ser Tyr Asp 15 69 7 PRT Artificial Sequence Description of Artificial SequenceProduct of synthesis based on mouse claudin-1 sequence 69 Lys Ile TyrSer Tyr Ala Asp 1 5 70 8 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on mouse claudin-1sequence 70 Lys Ile Tyr Ser Tyr Ala Gly Asp 1 5 71 7 PRT ArtificialSequence Description of Artificial Sequence Product of synthesis basedon mouse claudin-1 sequence 71 Lys Lys Ile Tyr Ser Tyr Asp 1 5 72 8 PRTArtificial Sequence Description of Artificial Sequence Product ofsynthesis based on mouse claudin-1 sequence 72 Lys Lys Ile Tyr Ser TyrAla Asp 1 5 73 9 PRT Artificial Sequence Description of ArtificialSequence Product of synthesis based on mouse claudin-1 sequence 73 LysLys Ile Tyr Ser Tyr Ala Gly Asp 1 5 74 8 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on mouseclaudin-1 sequence 74 Lys Trp Lys Ile Tyr Ser Tyr Asp 1 5 75 9 PRTArtificial Sequence Description of Artificial Sequence Product ofsynthesis based on mouse claudin-1 sequence 75 Lys Trp Lys Ile Tyr SerTyr Ala Asp 1 5 76 10 PRT Artificial Sequence Description of ArtificialSequence Product of synthesis based on mouse claudin-1 sequence 76 LysTrp Lys Ile Tyr Ser Tyr Ala Gly Asp 1 5 10 77 6 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on mouseclaudin-1 sequence 77 Lys Ile Tyr Ser Tyr Glu 1 5 78 7 PRT ArtificialSequence Description of Artificial Sequence Product of synthesis basedon mouse claudin-1 sequence 78 Lys Ile Tyr Ser Tyr Ala Glu 1 5 79 8 PRTArtificial Sequence Description of Artificial Sequence Product ofsynthesis based on mouse claudin-1 sequence 79 Lys Ile Tyr Ser Tyr AlaGly Glu 1 5 80 7 PRT Artificial Sequence Description of ArtificialSequence Product of synthesis based on mouse claudin-1 sequence 80 LysLys Ile Tyr Ser Tyr Glu 1 5 81 8 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on mouse claudin-1sequence 81 Lys Lys Ile Tyr Ser Tyr Ala Glu 1 5 82 9 PRT ArtificialSequence Description of Artificial Sequence Product of synthesis basedon mouse claudin-1 sequence 82 Lys Lys Ile Tyr Ser Tyr Ala Gly Glu 1 583 8 PRT Artificial Sequence Description of Artificial Sequence Productof synthesis based on mouse claudin-1 sequence 83 Lys Trp Lys Ile TyrSer Tyr Glu 1 5 84 9 PRT Artificial Sequence Description of ArtificialSequence Product of synthesis based on mouse claudin-1 sequence 84 LysTrp Lys Ile Tyr Ser Tyr Ala Glu 1 5 85 10 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on mouseclaudin-1 sequence 85 Lys Trp Lys Ile Tyr Ser Tyr Ala Gly Glu 1 5 10 866 PRT Artificial Sequence Description of Artificial Sequence Product ofsynthesis based on mouse claudin-1 sequence 86 Asp Ile Tyr Ser Tyr Lys 15 87 7 PRT Artificial Sequence Description of Artificial SequenceProduct of synthesis based on mouse claudin-1 sequence 87 Asp Ile TyrSer Tyr Ala Lys 1 5 88 8 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on mouse claudin-1sequence 88 Asp Ile Tyr Ser Tyr Ala Gly Lys 1 5 89 7 PRT ArtificialSequence Description of Artificial Sequence Product of synthesis basedon mouse claudin-1 sequence 89 Asp Lys Ile Tyr Ser Tyr Lys 1 5 90 8 PRTArtificial Sequence Description of Artificial Sequence Product ofsynthesis based on mouse claudin-1 sequence 90 Asp Lys Ile Tyr Ser TyrAla Lys 1 5 91 9 PRT Artificial Sequence Description of ArtificialSequence Product of synthesis based on mouse claudin-1 sequence 91 AspLys Ile Tyr Ser Tyr Ala Gly Lys 1 5 92 8 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on mouseclaudin-1 sequence 92 Asp Trp Lys Ile Tyr Ser Tyr Lys 1 5 93 9 PRTArtificial Sequence Description of Artificial Sequence Product ofsynthesis based on mouse claudin-1 sequence 93 Asp Trp Lys Ile Tyr SerTyr Ala Lys 1 5 94 10 PRT Artificial Sequence Description of ArtificialSequence Product of synthesis based on mouse claudin-1 sequence 94 AspTrp Lys Ile Tyr Ser Tyr Ala Gly Lys 1 5 10 95 6 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on mouseclaudin-1 sequence 95 Glu Ile Tyr Ser Tyr Lys 1 5 96 7 PRT ArtificialSequence Description of Artificial Sequence Product of synthesis basedon mouse claudin-1 sequence 96 Glu Ile Tyr Ser Tyr Ala Lys 1 5 97 8 PRTArtificial Sequence Description of Artificial Sequence Product ofsynthesis based on mouse claudin-1 sequence 97 Glu Ile Tyr Ser Tyr AlaGly Lys 1 5 98 7 PRT Artificial Sequence Description of ArtificialSequence Product of synthesis based on mouse claudin-1 sequence 98 GluLys Ile Tyr Ser Tyr Lys 1 5 99 8 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on mouse claudin-1sequence 99 Glu Lys Ile Tyr Ser Tyr Ala Lys 1 5 100 9 PRT ArtificialSequence Description of Artificial Sequence Product of synthesis basedon mouse claudin-1 sequence 100 Glu Lys Ile Tyr Ser Tyr Ala Gly Lys 1 5101 8 PRT Artificial Sequence Description of Artificial Sequence Productof synthesis based on mouse claudin-1 sequence 101 Glu Trp Lys Ile TyrSer Tyr Lys 1 5 102 9 PRT Artificial Sequence Description of ArtificialSequence Product of synthesis based on mouse claudin-1 sequence 102 GluTrp Lys Ile Tyr Ser Tyr Ala Lys 1 5 103 10 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on mouseclaudin-1 sequence 103 Glu Trp Lys Ile Tyr Ser Tyr Ala Gly Lys 1 5 10104 5 PRT Artificial Sequence Description of Artificial Sequence Productof synthesis based on mouse claudin-1 sequence 104 Ile Tyr Ser Tyr Ala 15 105 6 PRT Artificial Sequence Description of Artificial SequenceProduct of synthesis based on mouse claudin-1 sequence 105 Ile Tyr SerTyr Ala Gly 1 5 106 5 PRT Artificial Sequence Description of ArtificialSequence Product of synthesis based on mouse claudin-1 sequence 106 LysIle Tyr Ser Tyr 1 5 107 7 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on mouse claudin-1sequence 107 Lys Ile Tyr Ser Tyr Ala Gly 1 5 108 6 PRT ArtificialSequence Description of Artificial Sequence Product of synthesis basedon mouse claudin-1 sequence 108 Trp Lys Ile Tyr Ser Tyr 1 5 109 7 PRTArtificial Sequence Description of Artificial Sequence Product ofsynthesis based on mouse claudin-1 sequence 109 Trp Lys Ile Tyr Ser TyrAla 1 5 110 8 PRT Artificial Sequence Description of Artificial SequenceProduct of synthesis based on mouse claudin-1 sequence 110 Trp Lys IleTyr Ser Tyr Ala Gly 1 5 111 6 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on mouse claudin-2sequence 111 Cys Thr Ser Ser Tyr Cys 1 5 112 7 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on mouseclaudin-2 sequence 112 Cys Thr Ser Ser Tyr Val Cys 1 5 113 8 PRTArtificial Sequence Description of Artificial Sequence Product ofsynthesis based on mouse claudin-2 sequence 113 Cys Thr Ser Ser Tyr ValGly Cys 1 5 114 7 PRT Artificial Sequence Description of ArtificialSequence Product of synthesis based on mouse claudin-2 sequence 114 CysArg Thr Ser Ser Tyr Cys 1 5 115 8 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on mouse claudin-2sequence 115 Cys Arg Thr Ser Ser Tyr Val Cys 1 5 116 9 PRT ArtificialSequence Description of Artificial Sequence Product of synthesis basedon mouse claudin-2 sequence 116 Cys Arg Thr Ser Ser Tyr Val Gly Cys 1 5117 8 PRT Artificial Sequence Description of Artificial Sequence Productof synthesis based on mouse claudin-2 sequence 117 Cys Trp Arg Thr SerSer Tyr Cys 1 5 118 9 PRT Artificial Sequence Description of ArtificialSequence Product of synthesis based on mouse claudin-2 sequence 118 CysTrp Arg Thr Ser Ser Tyr Val Cys 1 5 119 10 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on mouseclaudin-2 sequence 119 Cys Trp Arg Thr Ser Ser Tyr Val Gly Cys 1 5 10120 6 PRT Artificial Sequence Description of Artificial Sequence Productof synthesis based on mouse claudin-2 sequence 120 Lys Thr Ser Ser TyrAsp 1 5 121 7 PRT Artificial Sequence Description of Artificial SequenceProduct of synthesis based on mouse claudin-2 sequence 121 Lys Thr ThrSer Tyr Val Asp 1 5 122 8 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on mouse claudin-2sequence 122 Lys Thr Thr Ser Tyr Val Gly Asp 1 5 123 7 PRT ArtificialSequence Description of Artificial Sequence Product of synthesis basedon mouse claudin-2 sequence 123 Lys Arg Thr Ser Ser Tyr Asp 1 5 124 8PRT Artificial Sequence Description of Artificial Sequence Product ofsynthesis based on mouse claudin-2 sequence 124 Lys Arg Thr Ser Ser TyrVal Asp 1 5 125 9 PRT Artificial Sequence Description of ArtificialSequence Product of synthesis based on mouse claudin-2 sequence 125 LysArg Thr Ser Ser Tyr Val Gly Asp 1 5 126 8 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on mouseclaudin-2 sequence 126 Lys Trp Arg Thr Ser Ser Tyr Asp 1 5 127 9 PRTArtificial Sequence Description of Artificial Sequence Product ofsynthesis based on mouse claudin-2 sequence 127 Lys Trp Arg Thr Ser SerTyr Val Asp 1 5 128 10 PRT Artificial Sequence Description of ArtificialSequence Product of synthesis based on mouse claudin-2 sequence 128 LysTrp Arg Thr Ser Ser Tyr Val Gly Asp 1 5 10 129 6 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on mouseclaudin-2 sequence 129 Lys Thr Ser Ser Tyr Glu 1 5 130 7 PRT ArtificialSequence Description of Artificial Sequence Product of synthesis basedon mouse claudin-2 sequence 130 Lys Thr Ser Ser Tyr Val Glu 1 5 131 8PRT Artificial Sequence Description of Artificial Sequence Product ofsynthesis based on mouse claudin-2 sequence 131 Lys Thr Ser Ser Tyr ValGly Glu 1 5 132 7 PRT Artificial Sequence Description of ArtificialSequence Product of synthesis based on mouse claudin-2 sequence 132 LysArg Thr Ser Ser Tyr Glu 1 5 133 8 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on mouse claudin-2sequence 133 Lys Arg Thr Ser Ser Tyr Val Glu 1 5 134 9 PRT ArtificialSequence Description of Artificial Sequence Product of synthesis basedon mouse claudin-2 sequence 134 Lys Arg Thr Ser Ser Tyr Val Gly Glu 1 5135 8 PRT Artificial Sequence Description of Artificial Sequence Productof synthesis based on mouse claudin-2 sequence 135 Lys Trp Arg Thr SerSer Tyr Glu 1 5 136 9 PRT Artificial Sequence Description of ArtificialSequence Product of synthesis based on mouse claudin-2 sequence 136 LysTrp Arg Thr Ser Ser Tyr Val Glu 1 5 137 10 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on mouseclaudin-2 sequence 137 Lys Trp Arg Thr Ser Ser Tyr Val Gly Glu 1 5 10138 6 PRT Artificial Sequence Description of Artificial Sequence Productof synthesis based on mouse claudin-2 sequence 138 Asp Thr Ser Ser TyrLys 1 5 139 7 PRT Artificial Sequence Description of Artificial SequenceProduct of synthesis based on mouse claudin-2 sequence 139 Asp Thr SerSer Tyr Val Lys 1 5 140 8 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on mouse claudin-2sequence 140 Asp Thr Ser Ser Tyr Val Gly Lys 1 5 141 7 PRT ArtificialSequence Description of Artificial Sequence Product of synthesis basedon mouse claudin-2 sequence 141 Asp Arg Thr Ser Ser Tyr Lys 1 5 142 8PRT Artificial Sequence Description of Artificial Sequence Product ofsynthesis based on mouse claudin-2 sequence 142 Asp Arg Thr Ser Ser TyrVal Lys 1 5 143 9 PRT Artificial Sequence Description of ArtificialSequence Product of synthesis based on mouse claudin-2 sequence 143 AspArg Thr Ser Ser Tyr Val Gly Lys 1 5 144 8 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on mouseclaudin-2 sequence 144 Asp Trp Arg Thr Ser Ser Tyr Lys 1 5 145 9 PRTArtificial Sequence Description of Artificial Sequence Product ofsynthesis based on mouse claudin-2 sequence 145 Asp Trp Arg Thr Ser SerTyr Val Lys 1 5 146 10 PRT Artificial Sequence Description of ArtificialSequence Product of synthesis based on mouse claudin-2 sequence 146 AspTrp Arg Thr Ser Ser Tyr Val Gly Lys 1 5 10 147 6 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on mouseclaudin-2 sequence 147 Glu Thr Ser Ser Tyr Lys 1 5 148 7 PRT ArtificialSequence Description of Artificial Sequence Product of synthesis basedon mouse claudin-2 sequence 148 Glu Thr Ser Ser Tyr Val Lys 1 5 149 8PRT Artificial Sequence Description of Artificial Sequence Product ofsynthesis based on mouse claudin-2 sequence 149 Glu Thr Thr Ser Tyr ValGly Lys 1 5 150 7 PRT Artificial Sequence Description of ArtificialSequence Product of synthesis based on mouse claudin-2 sequence 150 GluArg Thr Ser Ser Tyr Lys 1 5 151 8 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on mouse claudin-2sequence 151 Glu Arg Thr Ser Ser Tyr Val Lys 1 5 152 9 PRT ArtificialSequence Description of Artificial Sequence Product of synthesis basedon mouse claudin-2 sequence 152 Glu Arg Thr Ser Ser Tyr Val Gly Lys 1 5153 8 PRT Artificial Sequence Description of Artificial Sequence Productof synthesis based on mouse claudin-2 sequence 153 Glu Trp Arg Thr SerSer Tyr Lys 1 5 154 9 PRT Artificial Sequence Description of ArtificialSequence Product of synthesis based on mouse claudin-2 sequence 154 GluTrp Arg Thr Ser Ser Tyr Val Lys 1 5 155 10 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on mouseclaudin-2 sequence 155 Glu Trp Arg Thr Ser Ser Tyr Val Gly Lys 1 5 10156 5 PRT Artificial Sequence Description of Artificial Sequence Productof synthesis based on mouse claudin-2 sequence 156 Thr Ser Ser Tyr Val 15 157 6 PRT Artificial Sequence Description of Artificial SequenceProduct of synthesis based on mouse claudin-2 sequence 157 Thr Ser SerTyr Val Gly 1 5 158 5 PRT Artificial Sequence Description of ArtificialSequence Product of synthesis based on mouse claudin-2 sequence 158 ArgThr Ser Ser Tyr 1 5 159 6 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on mouse claudin-2sequence 159 Arg Thr Ser Ser Tyr Val 1 5 160 7 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on mouseclaudin-2 sequence 160 Arg Thr Ser Ser Tyr Val Gly 1 5 161 6 PRTArtificial Sequence Description of Artificial Sequence Product ofsynthesis based on mouse claudin-2 sequence 161 Trp Arg Thr Ser Ser Tyr1 5 162 7 PRT Artificial Sequence Description of Artificial SequenceProduct of synthesis based on mouse claudin-2 sequence 162 Trp Arg ThrSer Ser Tyr Val 1 5 163 8 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on mouse claudin-2sequence 163 Trp Arg Thr Ser Ser Tyr Val Gly 1 5 164 6 PRT ArtificialSequence Description of Artificial Sequence Product of synthesis basedon human, mouse and monkey CPE-R sequences 164 Cys Val Thr Ala Phe Cys 15 165 7 PRT Artificial Sequence Description of Artificial SequenceProduct of synthesis based on human, mouse and monkey CPE-R sequences165 Cys Val Thr Ala Phe Ile Cys 1 5 166 8 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on human,mouse and monkey CPE-R sequences 166 Cys Val Thr Ala Phe Ile Gly Cys 1 5167 7 PRT Artificial Sequence Description of Artificial Sequence Productof synthesis based on human, mouse and monkey CPE-R sequences 167 CysArg Val Thr Ala Phe Cys 1 5 168 8 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on human, mouse andmonkey CPE-R sequences 168 Cys Arg Val Thr Ala Phe Ile Cys 1 5 169 9 PRTArtificial Sequence Description of Artificial Sequence Product ofsynthesis based on human, mouse and monkey CPE-R sequences 169 Cys ArgVal Thr Ala Phe Ile Gly Cys 1 5 170 8 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on human,mouse and monkey CPE-R sequences 170 Cys Trp Arg Val Thr Ala Phe Cys 1 5171 9 PRT Artificial Sequence Description of Artificial Sequence Productof synthesis based on human, mouse and monkey CPE-R sequences 171 CysTrp Arg Val Thr Ala Phe Ile Cys 1 5 172 10 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on human,mouse and monkey CPE-R sequences 172 Cys Trp Arg Val Thr Ala Phe Ile GlyCys 1 5 10 173 6 PRT Artificial Sequence Description of ArtificialSequence Product of synthesis based on human, mouse and monkey CPE-Rsequences 173 Lys Val Thr Ala Phe Asp 1 5 174 7 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on human,mouse and monkey CPE-R sequences 174 Lys Val Thr Ala Phe Ile Asp 1 5 1758 PRT Artificial Sequence Description of Artificial Sequence Product ofsynthesis based on human, mouse and monkey CPE-R sequences 175 Lys ValThr Ala Phe Ile Gly Asp 1 5 176 7 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on human, mouse andmonkey CPE-R sequences 176 Lys Arg Val Thr Ala Phe Asp 1 5 177 8 PRTArtificial Sequence Description of Artificial Sequence Product ofsynthesis based on human, mouse and monkey CPE-R sequences 177 Lys ArgVal Thr Ala Phe Ile Asp 1 5 178 9 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on human, mouse andmonkey CPE-R sequences 178 Lys Arg Val Thr Ala Phe Ile Gly Asp 1 5 179 8PRT Artificial Sequence Description of Artificial Sequence Product ofsynthesis based on human, mouse and monkey CPE-R sequences 179 Lys TrpArg Val Thr Ala Phe Asp 1 5 180 9 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on human, mouse andmonkey CPE-R sequences 180 Lys Trp Arg Val Thr Ala Phe Ile Asp 1 5 18110 PRT Artificial Sequence Description of Artificial Sequence Product ofsynthesis based on human, mouse and monkey CPE-R sequences 181 Lys TrpArg Val Thr Ala Phe Ile Gly Asp 1 5 10 182 6 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on human,mouse and monkey CPE-R sequences 182 Lys Val Thr Ala Phe Glu 1 5 183 7PRT Artificial Sequence Description of Artificial Sequence Product ofsynthesis based on human, mouse and monkey CPE-R sequences 183 Lys ValThr Ala Phe Ile Glu 1 5 184 8 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on human, mouse andmonkey CPE-R sequences 184 Lys Val Thr Ala Phe Ile Gly Glu 1 5 185 7 PRTArtificial Sequence Description of Artificial Sequence Product ofsynthesis based on human, mouse and monkey CPE-R sequences 185 Lys ArgVal Thr Ala Phe Glu 1 5 186 8 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on human, mouse andmonkey CPE-R sequences 186 Lys Arg Val Thr Ala Phe Ile Glu 1 5 187 9 PRTArtificial Sequence Description of Artificial Sequence Product ofsynthesis based on human, mouse and monkey CPE-R sequences 187 Lys ArgVal Thr Ala Phe Ile Gly Glu 1 5 188 8 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on human,mouse and monkey CPE-R sequences 188 Lys Trp Arg Val Thr Ala Phe Glu 1 5189 9 PRT Artificial Sequence Description of Artificial Sequence Productof synthesis based on human, mouse and monkey CPE-R sequences 189 LysTrp Arg Val Thr Ala Phe Ile Glu 1 5 190 10 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on human,mouse and monkey CPE-R sequences 190 Lys Trp Arg Val Thr Ala Phe Ile GlyGlu 1 5 10 191 6 PRT Artificial Sequence Description of ArtificialSequence Product of synthesis based on human, mouse and monkey CPE-Rsequences 191 Asp Val Ala Thr Phe Lys 1 5 192 7 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on human,mouse and monkey CPE-R sequences 192 Asp Val Thr Ala Phe Ile Lys 1 5 1938 PRT Artificial Sequence Description of Artificial Sequence Product ofsynthesis based on human, mouse and monkey CPE-R sequences 193 Asp ValThr Ala Phe Ile Gly Lys 1 5 194 7 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on human, mouse andmonkey CPE-R sequences 194 Asp Arg Val Thr Ala Phe Lys 1 5 195 8 PRTArtificial Sequence Description of Artificial Sequence Product ofsynthesis based on human, mouse and monkey CPE-R sequences 195 Asp ArgVal Thr Ala Phe Ile Lys 1 5 196 9 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on human, mouse andmonkey CPE-R sequences 196 Asp Arg Val Thr Ala Phe Ile Gly Lys 1 5 197 8PRT Artificial Sequence Description of Artificial Sequence Product ofsynthesis based on human, mouse and monkey CPE-R sequences 197 Asp TrpArg Val Thr Ala Phe Lys 1 5 198 9 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on human, mouse andmonkey CPE-R sequences 198 Asp Trp Arg Val Thr Ala Phe Ile Lys 1 5 19910 PRT Artificial Sequence Description of Artificial Sequence Product ofsynthesis based on human, mouse and monkey CPE-R sequences 199 Asp TrpArg Val Thr Ala Phe Ile Gly Lys 1 5 10 200 6 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on human,mouse and monkey CPE-R sequences 200 Glu Val Thr Ala Phe Lys 1 5 201 7PRT Artificial Sequence Description of Artificial Sequence Product ofsynthesis based on human, mouse and monkey CPE-R sequences 201 Glu ValThr Ala Phe Ile Lys 1 5 202 8 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on human, mouse andmonkey CPE-R sequences 202 Glu Val Thr Ala Phe Ile Gly Lys 1 5 203 7 PRTArtificial Sequence Description of Artificial Sequence Product ofsynthesis based on human, mouse and monkey CPE-R sequences 203 Glu ArgVal Thr Ala Phe Lys 1 5 204 8 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on human, mouse andmonkey CPE-R sequences 204 Glu Arg Val Thr Ala Phe Ile Lys 1 5 205 9 PRTArtificial Sequence Description of Artificial Sequence Product ofsynthesis based on human, mouse and monkey CPE-R sequences 205 Glu ArgVal Thr Ala Phe Ile Gly Lys 1 5 206 8 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on human,mouse and monkey CPE-R sequences 206 Glu Trp Arg Val Thr Ala Phe Lys 1 5207 9 PRT Artificial Sequence Description of Artificial Sequence Productof synthesis based on human, mouse and monkey CPE-R sequences 207 GluTrp Arg Val Thr Ala Phe Ile Lys 1 5 208 10 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on human,mouse and monkey CPE-R sequences 208 Glu Trp Arg Val Thr Ala Phe Ile GlyLys 1 5 10 209 5 PRT Artificial Sequence Description of ArtificialSequence Product of synthesis based on human, mouse and monkey CPE-Rsequences 209 Val Thr Ala Phe Ile 1 5 210 6 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on human,mouse and monkey CPE-R sequences 210 Val Thr Ala Phe Ile Gly 1 5 211 5PRT Artificial Sequence Description of Artificial Sequence Product ofsynthesis based on human, mouse and monkey CPE-R sequences 211 Arg ValThr Ala Phe 1 5 212 6 PRT Artificial Sequence Description of ArtificialSequence Product of synthesis based on human, mouse and monkey CPE-Rsequences 212 Arg Val Thr Ala Phe Ile 1 5 213 7 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on human,mouse and monkey CPE-R sequences 213 Arg Val Thr Ala Phe Ile Gly 1 5 2146 PRT Artificial Sequence Description of Artificial Sequence Product ofsynthesis based on human, mouse and monkey CPE-R sequences 214 Trp ArgVal Thr Ala Phe 1 5 215 7 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on human, mouse andmonkey CPE-R sequences 215 Trp Arg Val Thr Ala Phe Ile 1 5 216 8 PRTArtificial Sequence Description of Artificial Sequence Product ofsynthesis based on human, mouse and monkey CPE-R sequences 216 Trp ArgVal Thr Ala Phe Ile Gly 1 5 217 6 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on human and rat RVP-1sequences 217 Cys Val Ser Ala Phe Cys 1 5 218 7 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on humanand rat RVP-1 sequences 218 Cys Val Ser Ala Phe Ile Cys 1 5 219 8 PRTArtificial Sequence Description of Artificial Sequence Product ofsynthesis based on human and rat RVP-1 sequences 219 Cys Val Ser Ala PheIle Gly Cys 1 5 220 7 PRT Artificial Sequence Description of ArtificialSequence Product of synthesis based on human and rat RVP-1 sequences 220Cys Arg Val Ser Ala Phe Cys 1 5 221 8 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on humanand rat RVP-1 sequences 221 Cys Arg Val Ser Ala Phe Ile Cys 1 5 222 9PRT Artificial Sequence Description of Artificial Sequence Product ofsynthesis based on human and rat RVP-1 sequences 222 Cys Arg Val Ser AlaPhe Ile Gly Cys 1 5 223 8 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on human and rat RVP-1sequences 223 Cys Trp Arg Val Ser Ala Phe Cys 1 5 224 9 PRT ArtificialSequence Description of Artificial Sequence Product of synthesis basedon human and rat RVP-1 sequences 224 Cys Trp Arg Val Ser Ala Phe Ile Cys1 5 225 10 PRT Artificial Sequence Description of Artificial SequenceProduct of synthesis based on human and rat RVP-1 sequences 225 Cys TrpArg Val Ser Ala Phe Ile Gly Cys 1 5 10 226 6 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on humanand rat RVP-1 sequences 226 Lys Val Ser Ala Phe Asp 1 5 227 7 PRTArtificial Sequence Description of Artificial Sequence Product ofsynthesis based on human and rat RVP-1 sequences 227 Lys Val Ser Ala PheIle Asp 1 5 228 8 PRT Artificial Sequence Description of ArtificialSequence Product of synthesis based on human and rat RVP-1 sequences 228Lys Val Ser Ala Phe Ile Gly Asp 1 5 229 7 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on humanand rat RVP-1 sequences 229 Lys Arg Val Ser Ala Phe Asp 1 5 230 8 PRTArtificial Sequence Description of Artificial Sequence Product ofsynthesis based on human and rat RVP-1 sequences 230 Lys Arg Val Ser AlaPhe Ile Asp 1 5 231 9 PRT Artificial Sequence Description of ArtificialSequence Product of synthesis based on human and rat RVP-1 sequences 231Lys Arg Val Ser Ala Phe Ile Gly Asp 1 5 232 8 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on humanand rat RVP-1 sequences 232 Lys Trp Arg Val Ser Ala Phe Asp 1 5 233 9PRT Artificial Sequence Description of Artificial Sequence Product ofsynthesis based on human and rat RVP-1 sequences 233 Lys Trp Arg Val SerAla Phe Ile Asp 1 5 234 10 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on human and rat RVP-1sequences 234 Lys Trp Arg Val Ser Ala Phe Ile Gly Asp 1 5 10 235 6 PRTArtificial Sequence Description of Artificial Sequence Product ofsynthesis based on human and rat RVP-1 sequences 235 Lys Val Ser Ala PheGlu 1 5 236 7 PRT Artificial Sequence Description of Artificial SequenceProduct of synthesis based on human and rat RVP-1 sequences 236 Lys ValSer Ala Phe Ile Glu 1 5 237 8 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on human and rat RVP-1sequences 237 Lys Val Ser Ala Phe Ile Gly Glu 1 5 238 7 PRT ArtificialSequence Description of Artificial Sequence Product of synthesis basedon human and rat RVP-1 sequences 238 Lys Arg Val Ser Ala Phe Glu 1 5 2398 PRT Artificial Sequence Description of Artificial Sequence Product ofsynthesis based on human and rat RVP-1 sequences 239 Lys Arg Val Ser AlaPhe Ile Glu 1 5 240 9 PRT Artificial Sequence Description of ArtificialSequence Product of synthesis based on human and rat RVP-1 sequences 240Lys Arg Val Ser Ala Phe Ile Gly Glu 1 5 241 8 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on humanand rat RVP-1 sequences 241 Lys Trp Arg Val Ser Ala Phe Glu 1 5 242 9PRT Artificial Sequence Description of Artificial Sequence Product ofsynthesis based on human and rat RVP-1 sequences 242 Lys Trp Arg Val SerAla Phe Ile Glu 1 5 243 10 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on human and rat RVP-1sequences 243 Lys Trp Arg Val Ser Ala Phe Ile Gly Glu 1 5 10 244 6 PRTArtificial Sequence Description of Artificial Sequence Product ofsynthesis based on human and rat RVP-1 sequences 244 Asp Val Ser Ala PheLys 1 5 245 7 PRT Artificial Sequence Description of Artificial SequenceProduct of synthesis based on human and rat RVP-1 sequences 245 Asp ValSer Ala Phe Ile Lys 1 5 246 8 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on human and rat RVP-1sequences 246 Asp Val Ser Ala Phe Ile Gly Lys 1 5 247 7 PRT ArtificialSequence Description of Artificial Sequence Product of synthesis basedon human and rat RVP-1 sequences 247 Asp Arg Val Ser Ala Phe Lys 1 5 2488 PRT Artificial Sequence Description of Artificial Sequence Product ofsynthesis based on human and rat RVP-1 sequences 248 Asp Arg Val Ser AlaPhe Ile Lys 1 5 249 9 PRT Artificial Sequence Description of ArtificialSequence Product of synthesis based on human and rat RVP-1 sequences 249Asp Arg Val Ser Ala Phe Ile Gly Lys 1 5 250 8 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on humanand rat RVP-1 sequences 250 Asp Trp Arg Val Ser Ala Phe Lys 1 5 251 9PRT Artificial Sequence Description of Artificial Sequence Product ofsynthesis based on human and rat RVP-1 sequences 251 Asp Trp Arg Val SerAla Phe Ile Lys 1 5 252 10 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on human and rat RVP-1sequences 252 Asp Trp Arg Val Ser Ala Phe Ile Gly Lys 1 5 10 253 6 PRTArtificial Sequence Description of Artificial Sequence Product ofsynthesis based on human and rat RVP-1 sequences 253 Glu Val Ser Ala PheLys 1 5 254 7 PRT Artificial Sequence Description of Artificial SequenceProduct of synthesis based on human and rat RVP-1 sequences 254 Glu ValSer Ala Phe Ile Lys 1 5 255 8 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on human and rat RVP-1sequences 255 Glu Val Ser Ala Phe Ile Gly Lys 1 5 256 7 PRT ArtificialSequence Description of Artificial Sequence Product of synthesis basedon human and rat RVP-1 sequences 256 Glu Arg Val Ser Ala Phe Lys 1 5 2578 PRT Artificial Sequence Description of Artificial Sequence Product ofsynthesis based on human and rat RVP-1 sequences 257 Glu Arg Val Ser AlaPhe Ile Lys 1 5 258 9 PRT Artificial Sequence Description of ArtificialSequence Product of synthesis based on human and rat RVP-1 sequences 258Glu Arg Val Ser Ala Phe Ile Gly Lys 1 5 259 8 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on humanand rat RVP-1 sequences 259 Glu Trp Arg Val Ser Ala Phe Lys 1 5 260 9PRT Artificial Sequence Description of Artificial Sequence Product ofsynthesis based on human and rat RVP-1 sequences 260 Glu Trp Arg Val SerAla Phe Ile Lys 1 5 261 10 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on human and rat RVP-1sequences 261 Glu Trp Arg Val Ser Ala Phe Ile Gly Lys 1 5 10 262 5 PRTArtificial Sequence Description of Artificial Sequence Product ofsynthesis based on human and rat RVP-1 sequences 262 Val Ser Ala Phe Ile1 5 263 6 PRT Artificial Sequence Description of Artificial SequenceProduct of synthesis based on human and rat RVP-1 sequences 263 Val SerAla Phe Ile Gly 1 5 264 5 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on human and rat RVP-1sequences 264 Arg Val Ser Ala Phe 1 5 265 6 PRT Artificial SequenceDescription of Artificial Sequence Product of synthesis based on humanand rat RVP-1 sequences 265 Arg Val Ser Ala Phe Ile 1 5 266 7 PRTArtificial Sequence Description of Artificial Sequence Product ofsynthesis based on human and rat RVP-1 sequences 266 Arg Val Ser Ala PheIle Gly 1 5 267 6 PRT Artificial Sequence Description of ArtificialSequence Product of synthesis based on human and rat RVP-1 sequences 267Trp Arg Val Ser Ala Phe 1 5 268 7 PRT Artificial Sequence Description ofArtificial Sequence Product of synthesis based on human and rat RVP-1sequences 268 Trp Arg Val Ser Ala Phe Ile 1 5 269 8 PRT ArtificialSequence Description of Artificial Sequence Product of synthesis basedon human and rat RVP-1 sequences 269 Trp Arg Val Ser Ala Phe Ile Gly 1 5270 5 PRT Artificial Sequence Representative linear modulating agentbased on claudin-5 cell adhesion recognition sequence 270 Val Thr AlaPhe Leu 1 5 271 6 PRT Artificial Sequence Representative linearmodulating agent based on claudin-5 cell adhesion recognition sequence271 Val Thr Ala Phe Leu Asp 1 5 272 5 PRT Artificial SequenceRepresentative linear modulating agent based on claudin-5 cell adhesionrecognition sequence 272 Gln Val Thr Ala Phe 1 5 273 6 PRT ArtificialSequence Representative linear modulating agent based on claudin-5 celladhesion recognition sequence 273 Gln Val Thr Ala Phe Leu 1 5 274 7 PRTArtificial Sequence Representative linear modulating agent based onclaudin-5 cell adhesion recognition sequence 274 Gln Val Thr Ala Phe LeuAsp 1 5 275 6 PRT Artificial Sequence Representative linear modulatingagent based on claudin-5 cell adhesion recognition sequence 275 Trp GlnVal Thr Ala Phe 1 5 276 7 PRT Artificial Sequence Representative linearmodulating agent based on claudin-5 cell adhesion recognition sequence276 Trp Gln Val Thr Ala Phe Leu 1 5 277 8 PRT Artificial SequenceRepresentative linear modulating agent based on claudin-5 cell adhesionrecognition sequence 277 Trp Gln Val Thr Ala Phe Leu Asp 1 5 278 7 PRTArtificial Sequence Representative cyclic modulating agent based onclaudin-5 cell adhesion recognition sequence 278 Cys Val Thr Ala Phe LeuCys 1 5 279 8 PRT Artificial Sequence Representative cyclic modulatingagent based on claudin-5 cell adhesion recognition sequence 279 Cys ValThr Ala Phe Leu Asp Cys 1 5 280 7 PRT Artificial Sequence Representativecyclic modulating agent based on claudin-5 cell adhesion recognitionsequence 280 Cys Gln Val Thr Ala Phe Cys 1 5 281 8 PRT ArtificialSequence Representative cyclic modulating agent based on claudin-5 celladhesion recognition sequence 281 Cys Gln Val Thr Ala Phe Leu Cys 1 5282 9 PRT Artificial Sequence Representative cyclic modulating agentbased on claudin-5 cell adhesion recognition sequence 282 Cys Gln ValThr Ala Phe Leu Asp Cys 1 5 283 8 PRT Artificial Sequence Representativecyclic modulating agent based on claudin-5 cell adhesion recognitionsequence 283 Cys Trp Gln Val Thr Ala Phe Cys 1 5 284 9 PRT ArtificialSequence Representative cyclic modulating agent based on claudin-5 celladhesion recognition sequence 284 Cys Trp Gln Val Thr Ala Phe Leu Cys 15 285 10 PRT Artificial Sequence Representative cyclic modulating agentbased on claudin-5 cell adhesion recognition sequence 285 Cys Trp GlnVal Thr Ala Phe Leu Asp Cys 1 5 10 286 6 PRT Artificial SequenceRepresentative cyclic modulating agent based on claudin-5 cell adhesionrecognition sequence 286 Lys Val Thr Ala Phe Asp 1 5 287 7 PRTArtificial Sequence Representative cyclic modulating agent based onclaudin-5 cell adhesion recognition sequence 287 Lys Val Thr Ala Phe LeuAsp 1 5 288 8 PRT Artificial Sequence Representative cyclic modulatingagent based on claudin-5 cell adhesion recognition sequence 288 Lys ValThr Ala Phe Leu Asp Asp 1 5 289 7 PRT Artificial Sequence Representativecyclic modulating agent based on claudin-5 cell adhesion recognitionsequence 289 Lys Gln Val Thr Ala Phe Asp 1 5 290 8 PRT ArtificialSequence Representative cyclic modulating agent based on claudin-5 celladhesion recognition sequence 290 Lys Gln Val Thr Ala Phe Leu Asp 1 5291 9 PRT Artificial Sequence Representative cyclic modulating agentbased on claudin-5 cell adhesion recognition sequence 291 Lys Gln ValThr Ala Phe Leu Asp Asp 1 5 292 8 PRT Artificial Sequence Representativecyclic modulating agent based on claudin-5 cell adhesion recognitionsequence 292 Lys Trp Gln Val Thr Ala Phe Asp 1 5 293 9 PRT ArtificialSequence Representative cyclic modulating agent based on claudin-5 celladhesion recognition sequence 293 Lys Trp Gln Val Thr Ala Phe Leu Asp 15 294 10 PRT Artificial Sequence Representative cyclic modulating agentbased on claudin-5 cell adhesion recognition sequence 294 Lys Trp GlnVal Thr Ala Phe Leu Asp Asp 1 5 10 295 7 PRT Artificial SequenceRepresentative cyclic modulating agent based on claudin-5 cell adhesionrecognition sequence 295 Lys Val Thr Ala Phe Leu Glu 1 5 296 8 PRTArtificial Sequence Representative cyclic modulating agent based onclaudin-5 cell adhesion recognition sequence 296 Lys Val Thr Ala Phe LeuAsp Glu 1 5 297 7 PRT Artificial Sequence Representative cyclicmodulating agent based on claudin-5 cell adhesion recognition sequence297 Lys Gln Val Thr Ala Phe Glu 1 5 298 8 PRT Artificial SequenceRepresentative cyclic modulating agent based on claudin-5 cell adhesionrecognition sequence 298 Lys Gln Val Thr Ala Phe Leu Glu 1 5 299 9 PRTArtificial Sequence Representative cyclic modulating agent based onclaudin-5 cell adhesion recognition sequence 299 Lys Gln Val Thr Ala PheLeu Asp Glu 1 5 300 8 PRT Artificial Sequence Representative cyclicmodulating agent based on claudin-5 cell adhesion recognition sequence300 Lys Trp Gln Val Thr Ala Phe Glu 1 5 301 9 PRT Artificial SequenceRepresentative cyclic modulating agent based on claudin-5 cell adhesionrecognition sequence 301 Lys Trp Gln Val Thr Ala Phe Leu Glu 1 5 302 10PRT Artificial Sequence Representative cyclic modulating agent based onclaudin-5 cell adhesion recognition sequence 302 Lys Trp Gln Val Thr AlaPhe Leu Asp Glu 1 5 10 303 6 PRT Artificial Sequence Representativecyclic modulating agent based on claudin-5 cell adhesion recognitionsequence 303 Asp Val Ala Thr Phe Lys 1 5 304 7 PRT Artificial SequenceRepresentative cyclic modulating agent based on claudin-5 cell adhesionrecognition sequence 304 Asp Val Thr Ala Phe Leu Lys 1 5 305 8 PRTArtificial Sequence Representative cyclic modulating agent based onclaudin-5 cell adhesion recognition sequence 305 Asp Val Thr Ala Phe LeuAsp Lys 1 5 306 7 PRT Artificial Sequence Representative cyclicmodulating agent based on claudin-5 cell adhesion recognition sequence306 Asp Gln Val Thr Ala Phe Lys 1 5 307 8 PRT Artificial SequenceRepresentative cyclic modulating agent based on claudin-5 cell adhesionrecognition sequence 307 Asp Gln Val Thr Ala Phe Leu Lys 1 5 308 9 PRTArtificial Sequence Representative cyclic modulating agent based onclaudin-5 cell adhesion recognition sequence 308 Asp Gln Val Thr Ala PheLeu Asp Lys 1 5 309 8 PRT Artificial Sequence Representative cyclicmodulating agent based on claudin-5 cell adhesion recognition sequence309 Asp Trp Gln Val Thr Ala Phe Lys 1 5 310 9 PRT Artificial SequenceRepresentative cyclic modulating agent based on claudin-5 cell adhesionrecognition sequence 310 Asp Trp Gln Val Thr Ala Phe Leu Lys 1 5 311 10PRT Artificial Sequence Representative cyclic modulating agent based onclaudin-5 cell adhesion recognition sequence 311 Asp Trp Gln Val Thr AlaPhe Leu Asp Lys 1 5 10 312 7 PRT Artificial Sequence Representativecyclic modulating agent based on claudin-5 cell adhesion recognitionsequence 312 Glu Val Thr Ala Phe Leu Lys 1 5 313 8 PRT ArtificialSequence Representative cyclic modulating agent based on claudin-5 celladhesion recognition sequence 313 Glu Val Thr Ala Phe Leu Asp Lys 1 5314 7 PRT Artificial Sequence Representative cyclic modulating agentbased on claudin-5 cell adhesion recognition sequence 314 Glu Gln ValThr Ala Phe Lys 1 5 315 8 PRT Artificial Sequence Representative cyclicmodulating agent based on claudin-5 cell adhesion recognition sequence315 Glu Gln Val Thr Ala Phe Leu Lys 1 5 316 9 PRT Artificial SequenceRepresentative cyclic modulating agent based on claudin-5 cell adhesionrecognition sequence 316 Glu Gln Val Thr Ala Phe Leu Asp Lys 1 5 317 8PRT Artificial Sequence Representative cyclic modulating agent based onclaudin-5 cell adhesion recognition sequence 317 Glu Trp Gln Val Thr AlaPhe Lys 1 5 318 9 PRT Artificial Sequence Representative cyclicmodulating agent based on claudin-5 cell adhesion recognition sequence318 Glu Trp Gln Val Thr Ala Phe Leu Lys 1 5 319 10 PRT ArtificialSequence Representative cyclic modulating agent based on claudin-5 celladhesion recognition sequence 319 Glu Trp Gln Val Thr Ala Phe Leu AspLys 1 5 10 320 5 PRT Artificial Sequence Representative cyclicmodulating agent based on claudin-5 cell adhesion recognition sequence320 Val Thr Ala Phe Leu 1 5 321 6 PRT Artificial Sequence Representativecyclic modulating agent based on claudin-5 cell adhesion recognitionsequence 321 Val Thr Ala Phe Leu Asp 1 5 322 5 PRT Artificial SequenceRepresentative cyclic modulating agent based on claudin-5 cell adhesionrecognition sequence 322 Gln Val Thr Ala Phe 1 5 323 6 PRT ArtificialSequence Representative cyclic modulating agent based on claudin-5 celladhesion recognition sequence 323 Gln Val Thr Ala Phe Leu 1 5 324 7 PRTArtificial Sequence Representative cyclic modulating agent based onclaudin-5 cell adhesion recognition sequence 324 Gln Val Thr Ala Phe LeuAsp 1 5 325 6 PRT Artificial Sequence Representative cyclic modulatingagent based on claudin-5 cell adhesion recognition sequence 325 Trp GlnVal Thr Ala Phe 1 5 326 7 PRT Artificial Sequence Representative cyclicmodulating agent based on claudin-5 cell adhesion recognition sequence326 Trp Gln Val Thr Ala Phe Leu 1 5 327 8 PRT Artificial SequenceRepresentative cyclic modulating agent based on claudin-5 cell adhesionrecognition sequence 327 Trp Gln Val Thr Ala Phe Leu Asp 1 5 328 5 PRTArtificial Sequence Representative linear modulating agent based onclaudin-6 cell adhesion recognition sequence 328 Val Thr Ala Phe Ile 1 5329 6 PRT Artificial Sequence Representative linear modulating agentbased on claudin-6 cell adhesion recognition sequence 329 Val Thr AlaPhe Ile Gly 1 5 330 5 PRT Artificial Sequence Representative linearmodulating agent based on claudin-6 cell adhesion recognition sequence330 Lys Val Thr Ala Phe 1 5 331 6 PRT Artificial Sequence Representativelinear modulating agent based on claudin-6 cell adhesion recognitionsequence 331 Lys Val Thr Ala Phe Ile 1 5 332 7 PRT Artificial SequenceRepresentative linear modulating agent based on claudin-6 cell adhesionrecognition sequence 332 Lys Val Thr Ala Phe Ile Gly 1 5 333 6 PRTArtificial Sequence Representative linear modulating agent based onclaudin-6 cell adhesion recognition sequence 333 Trp Lys Val Thr Ala Phe1 5 334 7 PRT Artificial Sequence Representative linear modulating agentbased on claudin-6 cell adhesion recognition sequence 334 Trp Lys ValThr Ala Phe Ile 1 5 335 8 PRT Artificial Sequence Representative linearmodulating agent based on claudin-6 cell adhesion recognition sequence335 Trp Lys Val Thr Ala Phe Ile Gly 1 5 336 7 PRT Artificial SequenceRepresentative cyclic modulating agent based on claudin-6 cell adhesionrecognition sequence 336 Cys Val Thr Ala Phe Ile Cys 1 5 337 8 PRTArtificial Sequence Representative cyclic modulating agent based onclaudin-6 cell adhesion recognition sequence 337 Cys Val Thr Ala Phe IleGly Cys 1 5 338 7 PRT Artificial Sequence Representative cyclicmodulating agent based on claudin-6 cell adhesion recognition sequence338 Cys Lys Val Thr Ala Phe Cys 1 5 339 8 PRT Artificial SequenceRepresentative cyclic modulating agent based on claudin-6 cell adhesionrecognition sequence 339 Cys Lys Val Thr Ala Phe Ile Cys 1 5 340 9 PRTArtificial Sequence Representative cyclic modulating agent based onclaudin-6 cell adhesion recognition sequence 340 Cys Lys Val Thr Ala PheIle Gly Cys 1 5 341 8 PRT Artificial Sequence Representative cyclicmodulating agent based on claudin-6 cell adhesion recognition sequence341 Cys Trp Lys Val Thr Ala Phe Cys 1 5 342 9 PRT Artificial SequenceRepresentative cyclic modulating agent based on claudin-6 cell adhesionrecognition sequence 342 Cys Trp Lys Val Thr Ala Phe Ile Cys 1 5 343 10PRT Artificial Sequence Representative cyclic modulating agent based onclaudin-6 cell adhesion recognition sequence 343 Cys Trp Lys Val Thr AlaPhe Ile Gly Cys 1 5 10 344 6 PRT Artificial Sequence Representativecyclic modulating agent based on claudin-6 cell adhesion recognitionsequence 344 Lys Val Thr Ala Phe Asp 1 5 345 7 PRT Artificial SequenceRepresentative cyclic modulating agent based on claudin-6 cell adhesionrecognition sequence 345 Lys Val Thr Ala Phe Ile Asp 1 5 346 8 PRTArtificial Sequence Representative cyclic modulating agent based onclaudin-6 cell adhesion recognition sequence 346 Lys Val Thr Ala Phe IleGly Asp 1 5 347 7 PRT Artificial Sequence Representative cyclicmodulating agent based on claudin-6 cell adhesion recognition sequence347 Lys Lys Val Thr Ala Phe Asp 1 5 348 8 PRT Artificial SequenceRepresentative cyclic modulating agent based on claudin-6 cell adhesionrecognition sequence 348 Lys Lys Val Thr Ala Phe Ile Asp 1 5 349 9 PRTArtificial Sequence Representative cyclic modulating agent based onclaudin-6 cell adhesion recognition sequence 349 Lys Lys Val Thr Ala PheIle Gly Asp 1 5 350 8 PRT Artificial Sequence Representative cyclicmodulating agent based on claudin-6 cell adhesion recognition sequence350 Lys Trp Lys Val Thr Ala Phe Asp 1 5 351 9 PRT Artificial SequenceRepresentative cyclic modulating agent based on claudin-6 cell adhesionrecognition sequence 351 Lys Trp Lys Val Thr Ala Phe Ile Asp 1 5 352 10PRT Artificial Sequence Representative cyclic modulating agent based onclaudin-6 cell adhesion recognition sequence 352 Lys Trp Lys Val Thr AlaPhe Ile Gly Asp 1 5 10 353 7 PRT Artificial Sequence Representativecyclic modulating agent based on claudin-6 cell adhesion recognitionsequence 353 Lys Val Thr Ala Phe Ile Glu 1 5 354 8 PRT ArtificialSequence Representative cyclic modulating agent based on claudin-6 celladhesion recognition sequence 354 Lys Val Thr Ala Phe Ile Gly Glu 1 5355 7 PRT Artificial Sequence Representative cyclic modulating agentbased on claudin-6 cell adhesion recognition sequence 355 Lys Lys ValThr Ala Phe Glu 1 5 356 8 PRT Artificial Sequence Representative cyclicmodulating agent based on claudin-6 cell adhesion recognition sequence356 Lys Lys Val Thr Ala Phe Ile Glu 1 5 357 9 PRT Artificial SequenceRepresentative cyclic modulating agent based on claudin-6 cell adhesionrecognition sequence 357 Lys Lys Val Thr Ala Phe Ile Gly Glu 1 5 358 8PRT Artificial Sequence Representative cyclic modulating agent based onclaudin-6 cell adhesion recognition sequence 358 Lys Trp Lys Val Thr AlaPhe Glu 1 5 359 9 PRT Artificial Sequence Representative cyclicmodulating agent based on claudin-6 cell adhesion recognition sequence359 Lys Trp Lys Val Thr Ala Phe Ile Glu 1 5 360 10 PRT ArtificialSequence Representative cyclic modulating agent based on claudin-6 celladhesion recognition sequence 360 Lys Trp Lys Val Thr Ala Phe Ile GlyGlu 1 5 10 361 6 PRT Artificial Sequence Representative cyclicmodulating agent based on claudin-6 cell adhesion recognition sequence361 Asp Val Ala Thr Phe Lys 1 5 362 7 PRT Artificial SequenceRepresentative cyclic modulating agent based on claudin-6 cell adhesionrecognition sequence 362 Asp Val Thr Ala Phe Ile Lys 1 5 363 8 PRTArtificial Sequence Representative cyclic modulating agent based onclaudin-6 cell adhesion recognition sequence 363 Asp Val Thr Ala Phe IleGly Lys 1 5 364 7 PRT Artificial Sequence Representative cyclicmodulating agent based on claudin-6 cell adhesion recognition sequence364 Asp Lys Val Thr Ala Phe Lys 1 5 365 8 PRT Artificial SequenceRepresentative cyclic modulating agent based on claudin-6 cell adhesionrecognition sequence 365 Asp Lys Val Thr Ala Phe Ile Lys 1 5 366 9 PRTArtificial Sequence Representative cyclic modulating agent based onclaudin-6 cell adhesion recognition sequence 366 Asp Lys Val Thr Ala PheIle Gly Lys 1 5 367 8 PRT Artificial Sequence Representative cyclicmodulating agent based on claudin-6 cell adhesion recognition sequence367 Asp Trp Lys Val Thr Ala Phe Lys 1 5 368 9 PRT Artificial SequenceRepresentative cyclic modulating agent based on claudin-6 cell adhesionrecognition sequence 368 Asp Trp Lys Val Thr Ala Phe Ile Lys 1 5 369 10PRT Artificial Sequence Representative cyclic modulating agent based onclaudin-6 cell adhesion recognition sequence 369 Asp Trp Lys Val Thr AlaPhe Ile Gly Lys 1 5 10 370 7 PRT Artificial Sequence Representativecyclic modulating agent based on claudin-6 cell adhesion recognitionsequence 370 Glu Val Thr Ala Phe Ile Lys 1 5 371 8 PRT ArtificialSequence Representative cyclic modulating agent based on claudin-6 celladhesion recognition sequence 371 Glu Val Thr Ala Phe Ile Gly Lys 1 5372 7 PRT Artificial Sequence Representative cyclic modulating agentbased on claudin-6 cell adhesion recognition sequence 372 Glu Lys ValThr Ala Phe Lys 1 5 373 8 PRT Artificial Sequence Representative cyclicmodulating agent based on claudin-6 cell adhesion recognition sequence373 Glu Lys Val Thr Ala Phe Ile Lys 1 5 374 9 PRT Artificial SequenceRepresentative cyclic modulating agent based on claudin-6 cell adhesionrecognition sequence 374 Glu Lys Val Thr Ala Phe Ile Gly Lys 1 5 375 8PRT Artificial Sequence Representative cyclic modulating agent based onclaudin-6 cell adhesion recognition sequence 375 Glu Trp Lys Val Thr AlaPhe Lys 1 5 376 9 PRT Artificial Sequence Representative cyclicmodulating agent based on claudin-6 cell adhesion recognition sequence376 Glu Trp Lys Val Thr Ala Phe Ile Lys 1 5 377 10 PRT ArtificialSequence Representative cyclic modulating agent based on claudin-6 celladhesion recognition sequence 377 Glu Trp Lys Val Thr Ala Phe Ile GlyLys 1 5 10 378 5 PRT Artificial Sequence Representative cyclicmodulating agent based on claudin-6 cell adhesion recognition sequence378 Val Thr Ala Phe Ile 1 5 379 6 PRT Artificial Sequence Representativecyclic modulating agent based on claudin-6 cell adhesion recognitionsequence 379 Val Thr Ala Phe Ile Gly 1 5 380 5 PRT Artificial SequenceRepresentative cyclic modulating agent based on claudin-6 cell adhesionrecognition sequence 380 Lys Val Thr Ala Phe 1 5 381 6 PRT ArtificialSequence Representative cyclic modulating agent based on claudin-6 celladhesion recognition sequence 381 Lys Val Thr Ala Phe Ile 1 5 382 7 PRTArtificial Sequence Representative cyclic modulating agent based onclaudin-6 cell adhesion recognition sequence 382 Lys Val Thr Ala Phe IleGly 1 5 383 6 PRT Artificial Sequence Representative cyclic modulatingagent based on claudin-6 cell adhesion recognition sequence 383 Trp LysVal Thr Ala Phe 1 5 384 7 PRT Artificial Sequence Representative cyclicmodulating agent based on claudin-6 cell adhesion recognition sequence384 Trp Lys Val Thr Ala Phe Ile 1 5 385 8 PRT Artificial SequenceRepresentative cyclic modulating agent based on claudin-6 cell adhesionrecognition sequence 385 Trp Lys Val Thr Ala Phe Ile Gly 1 5 386 4 PRTArtificial Sequence Representative linear modulating agent based onclaudin-7 cell adhesion recognition sequence 386 Met Ser Ser Tyr 1 387 5PRT Artificial Sequence Representative linear modulating agent based onclaudin-7 cell adhesion recognition sequence 387 Met Ser Ser Tyr Ala 1 5388 6 PRT Artificial Sequence Representative linear modulating agentbased on claudin-7 cell adhesion recognition sequence 388 Met Ser SerTyr Ala Gly 1 5 389 5 PRT Artificial Sequence Representative linearmodulating agent based on claudin-7 cell adhesion recognition sequence389 Gln Met Ser Ser Tyr 1 5 390 6 PRT Artificial Sequence Representativelinear modulating agent based on claudin-7 cell adhesion recognitionsequence 390 Gln Met Ser Ser Tyr Ala 1 5 391 7 PRT Artificial SequenceRepresentative linear modulating agent based on claudin-7 cell adhesionrecognition sequence 391 Gln Met Ser Ser Tyr Ala Gly 1 5 392 6 PRTArtificial Sequence Representative linear modulating agent based onclaudin-7 cell adhesion recognition sequence 392 Trp Gln Met Ser Ser Tyr1 5 393 7 PRT Artificial Sequence Representative linear modulating agentbased on claudin-7 cell adhesion recognition sequence 393 Trp Gln MetSer Ser Tyr Ala 1 5 394 8 PRT Artificial Sequence Representative linearmodulating agent based on claudin-7 cell adhesion recognition sequence394 Trp Gln Met Ser Ser Tyr Ala Gly 1 5 395 6 PRT Artificial SequenceRepresentative linear modulating agent based on claudin-7 cell adhesionrecognition sequence 395 Cys Met Ser Ser Tyr Cys 1 5 396 7 PRTArtificial Sequence Representative linear modulating agent based onclaudin-7 cell adhesion recognition sequence 396 Cys Met Ser Ser Tyr AlaCys 1 5 397 8 PRT Artificial Sequence Representative linear modulatingagent based on claudin-7 cell adhesion recognition sequence 397 Cys MetSer Ser Tyr Ala Gly Cys 1 5 398 7 PRT Artificial Sequence Representativelinear modulating agent based on claudin-7 cell adhesion recognitionsequence 398 Cys Gln Met Ser Ser Tyr Cys 1 5 399 8 PRT ArtificialSequence Representative linear modulating agent based on claudin-7 celladhesion recognition sequence 399 Cys Gln Met Ser Ser Tyr Ala Cys 1 5400 9 PRT Artificial Sequence Representative linear modulating agentbased on claudin-7 cell adhesion recognition sequence 400 Cys Gln MetSer Ser Tyr Ala Gly Cys 1 5 401 8 PRT Artificial Sequence Representativelinear modulating agent based on claudin-7 cell adhesion recognitionsequence 401 Cys Trp Gln Met Ser Ser Tyr Cys 1 5 402 9 PRT ArtificialSequence Representative linear modulating agent based on claudin-7 celladhesion recognition sequence 402 Cys Trp Gln Met Ser Ser Tyr Ala Cys 15 403 10 PRT Artificial Sequence Representative linear modulating agentbased on claudin-7 cell adhesion recognition sequence 403 Cys Trp GlnMet Ser Ser Tyr Ala Gly Cys 1 5 10 404 6 PRT Artificial SequenceRepresentative linear modulating agent based on claudin-7 cell adhesionrecognition sequence 404 Lys Met Ser Ser Tyr Asp 1 5 405 7 PRTArtificial Sequence Representative linear modulating agent based onclaudin-7 cell adhesion recognition sequence 405 Lys Met Ser Ser Tyr AlaAsp 1 5 406 8 PRT Artificial Sequence Representative linear modulatingagent based on claudin-7 cell adhesion recognition sequence 406 Lys MetSer Ser Tyr Ala Gly Asp 1 5 407 7 PRT Artificial Sequence Representativelinear modulating agent based on claudin-7 cell adhesion recognitionsequence 407 Lys Gln Met Ser Ser Tyr Asp 1 5 408 8 PRT ArtificialSequence Representative linear modulating agent based on claudin-7 celladhesion recognition sequence 408 Lys Gln Met Ser Ser Tyr Ala Asp 1 5409 9 PRT Artificial Sequence Representative linear modulating agentbased on claudin-7 cell adhesion recognition sequence 409 Lys Gln MetSer Ser Tyr Ala Gly Asp 1 5 410 8 PRT Artificial Sequence Representativelinear modulating agent based on claudin-7 cell adhesion recognitionsequence 410 Lys Trp Gln Met Ser Ser Tyr Asp 1 5 411 9 PRT ArtificialSequence Representative linear modulating agent based on claudin-7 celladhesion recognition sequence 411 Lys Trp Gln Met Ser Ser Tyr Ala Asp 15 412 10 PRT Artificial Sequence Representative linear modulating agentbased on claudin-7 cell adhesion recognition sequence 412 Lys Trp GlnMet Ser Ser Tyr Ala Gly Asp 1 5 10 413 6 PRT Artificial SequenceRepresentative linear modulating agent based on claudin-7 cell adhesionrecognition sequence 413 Lys Met Ser Ser Tyr Glu 1 5 414 7 PRTArtificial Sequence Representative linear modulating agent based onclaudin-7 cell adhesion recognition sequence 414 Lys Met Ser Ser Tyr AlaGlu 1 5 415 8 PRT Artificial Sequence Representative linear modulatingagent based on claudin-7 cell adhesion recognition sequence 415 Lys MetSer Ser Tyr Ala Gly Glu 1 5 416 7 PRT Artificial Sequence Representativelinear modulating agent based on claudin-7 cell adhesion recognitionsequence 416 Lys Gln Met Ser Ser Tyr Glu 1 5 417 8 PRT ArtificialSequence Representative linear modulating agent based on claudin-7 celladhesion recognition sequence 417 Lys Gln Met Ser Ser Tyr Ala Glu 1 5418 9 PRT Artificial Sequence Representative linear modulating agentbased on claudin-7 cell adhesion recognition sequence 418 Lys Gln MetSer Ser Tyr Ala Gly Glu 1 5 419 8 PRT Artificial Sequence Representativelinear modulating agent based on claudin-7 cell adhesion recognitionsequence 419 Lys Trp Gln Met Ser Ser Tyr Glu 1 5 420 9 PRT ArtificialSequence Representative linear modulating agent based on claudin-7 celladhesion recognition sequence 420 Lys Trp Gln Met Ser Ser Tyr Ala Glu 15 421 10 PRT Artificial Sequence Representative linear modulating agentbased on claudin-7 cell adhesion recognition sequence 421 Lys Trp GlnMet Ser Ser Tyr Ala Gly Glu 1 5 10 422 6 PRT Artificial SequenceRepresentative linear modulating agent based on claudin-7 cell adhesionrecognition sequence 422 Asp Met Ser Ser Tyr Lys 1 5 423 7 PRTArtificial Sequence Representative linear modulating agent based onclaudin-7 cell adhesion recognition sequence 423 Asp Met Ser Ser Tyr AlaLys 1 5 424 8 PRT Artificial Sequence Representative linear modulatingagent based on claudin-7 cell adhesion recognition sequence 424 Asp MetSer Ser Tyr Ala Gly Lys 1 5 425 7 PRT Artificial Sequence Representativelinear modulating agent based on claudin-7 cell adhesion recognitionsequence 425 Asp Gln Met Ser Ser Tyr Lys 1 5 426 8 PRT ArtificialSequence Representative linear modulating agent based on claudin-7 celladhesion recognition sequence 426 Asp Gln Met Ser Ser Tyr Ala Lys 1 5427 9 PRT Artificial Sequence Representative linear modulating agentbased on claudin-7 cell adhesion recognition sequence 427 Asp Gln MetSer Ser Tyr Ala Gly Lys 1 5 428 8 PRT Artificial Sequence Representativelinear modulating agent based on claudin-7 cell adhesion recognitionsequence 428 Asp Trp Gln Met Ser Ser Tyr Lys 1 5 429 9 PRT ArtificialSequence Representative linear modulating agent based on claudin-7 celladhesion recognition sequence 429 Asp Trp Gln Met Ser Ser Tyr Ala Lys 15 430 10 PRT Artificial Sequence Representative linear modulating agentbased on claudin-7 cell adhesion recognition sequence 430 Asp Trp GlnMet Ser Ser Tyr Ala Gly Lys 1 5 10 431 6 PRT Artificial SequenceRepresentative linear modulating agent based on claudin-7 cell adhesionrecognition sequence 431 Glu Met Ser Ser Tyr Lys 1 5 432 7 PRTArtificial Sequence Representative linear modulating agent based onclaudin-7 cell adhesion recognition sequence 432 Glu Met Ser Ser Tyr AlaLys 1 5 433 8 PRT Artificial Sequence Representative linear modulatingagent based on claudin-7 cell adhesion recognition sequence 433 Glu MetSer Ser Tyr Ala Gly Lys 1 5 434 7 PRT Artificial Sequence Representativelinear modulating agent based on claudin-7 cell adhesion recognitionsequence 434 Glu Gln Met Ser Ser Tyr Lys 1 5 435 8 PRT ArtificialSequence Representative linear modulating agent based on claudin-7 celladhesion recognition sequence 435 Glu Gln Met Ser Ser Tyr Ala Lys 1 5436 9 PRT Artificial Sequence Representative linear modulating agentbased on claudin-7 cell adhesion recognition sequence 436 Glu Gln MetSer Ser Tyr Ala Gly Lys 1 5 437 8 PRT Artificial Sequence Representativelinear modulating agent based on claudin-7 cell adhesion recognitionsequence 437 Glu Trp Gln Met Ser Ser Tyr Lys 1 5 438 9 PRT ArtificialSequence Representative linear modulating agent based on claudin-7 celladhesion recognition sequence 438 Glu Trp Gln Met Ser Ser Tyr Ala Lys 15 439 10 PRT Artificial Sequence Representative linear modulating agentbased on claudin-7 cell adhesion recognition sequence 439 Glu Trp GlnMet Ser Ser Tyr Ala Gly Lys 1 5 10 440 5 PRT Artificial SequenceRepresentative linear modulating agent based on claudin-7 cell adhesionrecognition sequence 440 Met Ser Ser Tyr Ala 1 5 441 6 PRT ArtificialSequence Representative linear modulating agent based on claudin-7 celladhesion recognition sequence 441 Met Ser Ser Tyr Ala Gly 1 5 442 5 PRTArtificial Sequence Representative linear modulating agent based onclaudin-7 cell adhesion recognition sequence 442 Gln Met Ser Ser Tyr 1 5443 6 PRT Artificial Sequence Representative linear modulating agentbased on claudin-7 cell adhesion recognition sequence 443 Gln Met SerSer Tyr Ala 1 5 444 7 PRT Artificial Sequence Representative linearmodulating agent based on claudin-7 cell adhesion recognition sequence444 Gln Met Ser Ser Tyr Ala Gly 1 5 445 6 PRT Artificial SequenceRepresentative linear modulating agent based on claudin-7 cell adhesionrecognition sequence 445 Trp Gln Met Ser Ser Tyr 1 5 446 7 PRTArtificial Sequence Representative linear modulating agent based onclaudin-7 cell adhesion recognition sequence 446 Trp Gln Met Ser Ser TyrAla 1 5 447 8 PRT Artificial Sequence Representative linear modulatingagent based on claudin-7 cell adhesion recognition sequence 447 Trp GlnMet Ser Ser Tyr Ala Gly 1 5 448 6 PRT Artificial Sequence Representativelinear modulating agent based on claudin-8 cell adhesion recognitionsequence 448 Val Ser Ala Phe Ile Glu 1 5 449 7 PRT Artificial SequenceRepresentative linear modulating agent based on claudin-8 cell adhesionrecognition sequence 449 Arg Val Ser Ala Phe Ile Glu 1 5 450 8 PRTArtificial Sequence Representative linear modulating agent based onclaudin-8 cell adhesion recognition sequence 450 Trp Arg Val Ser Ala PheIle Glu 1 5 451 8 PRT Artificial Sequence Representative cyclicmodulating agent based on claudin-8 cell adhesion recognition sequence451 Cys Val Ser Ala Phe Ile Glu Cys 1 5 452 9 PRT Artificial SequenceRepresentative cyclic modulating agent based on claudin-8 cell adhesionrecognition sequence 452 Cys Arg Val Ser Ala Phe Ile Glu Cys 1 5 453 10PRT Artificial Sequence Representative cyclic modulating agent based onclaudin-8 cell adhesion recognition sequence 453 Cys Trp Arg Val Ser AlaPhe Ile Glu Cys 1 5 10 454 8 PRT Artificial Sequence Representativecyclic modulating agent based on claudin-8 cell adhesion recognitionsequence 454 Lys Val Ser Ala Phe Ile Glu Asp 1 5 455 9 PRT ArtificialSequence Representative cyclic modulating agent based on claudin-8 celladhesion recognition sequence 455 Lys Arg Val Ser Ala Phe Ile Glu Asp 15 456 10 PRT Artificial Sequence Representative cyclic modulating agentbased on claudin-8 cell adhesion recognition sequence 456 Lys Trp ArgVal Ser Ala Phe Ile Glu Asp 1 5 10 457 8 PRT Artificial SequenceRepresentative cyclic modulating agent based on claudin-8 cell adhesionrecognition sequence 457 Lys Val Ser Ala Phe Ile Glu Glu 1 5 458 9 PRTArtificial Sequence Representative cyclic modulating agent based onclaudin-8 cell adhesion recognition sequence 458 Lys Arg Val Ser Ala PheIle Glu Glu 1 5 459 10 PRT Artificial Sequence Representative cyclicmodulating agent based on claudin-8 cell adhesion recognition sequence459 Lys Trp Arg Val Ser Ala Phe Ile Glu Glu 1 5 10 460 8 PRT ArtificialSequence Representative cyclic modulating agent based on claudin-8 celladhesion recognition sequence 460 Asp Val Ser Ala Phe Ile Glu Lys 1 5461 9 PRT Artificial Sequence Representative cyclic modulating agentbased on claudin-8 cell adhesion recognition sequence 461 Asp Arg ValSer Ala Phe Ile Glu Lys 1 5 462 10 PRT Artificial SequenceRepresentative cyclic modulating agent based on claudin-8 cell adhesionrecognition sequence 462 Asp Trp Arg Val Ser Ala Phe Ile Glu Lys 1 5 10463 8 PRT Artificial Sequence Representative cyclic modulating agentbased on claudin-8 cell adhesion recognition sequence 463 Glu Val SerAla Phe Ile Glu Lys 1 5 464 9 PRT Artificial Sequence Representativecyclic modulating agent based on claudin-8 cell adhesion recognitionsequence 464 Glu Arg Val Ser Ala Phe Ile Glu Lys 1 5 465 10 PRTArtificial Sequence Representative cyclic modulating agent based onclaudin-8 cell adhesion recognition sequence 465 Glu Trp Arg Val Ser AlaPhe Ile Glu Lys 1 5 10 466 6 PRT Artificial Sequence Representativecyclic modulating agent based on claudin-8 cell adhesion recognitionsequence 466 Val Ser Ala Phe Ile Glu 1 5 467 7 PRT Artificial SequenceRepresentative cyclic modulating agent based on claudin-8 cell adhesionrecognition sequence 467 Arg Val Ser Ala Phe Ile Glu 1 5 468 8 PRTArtificial Sequence Representative cyclic modulating agent based onclaudin-8 cell adhesion recognition sequence 468 Trp Arg Val Ser Ala PheIle Glu 1 5 469 30 PRT Mus musculus 469 Pro Met Trp Arg Val Ser Ala PheIle Gly Ser Ser Ile Ile Thr Ala 1 5 10 15 Gln Ile Thr Trp Glu Gly LeuTrp Met Asn Cys Val Val Gln 20 25 30 470 30 PRT Mus musculus 470 Pro MetTrp Gln Val Thr Ala Phe Leu Asp His Asn Ile Val Thr Ala 1 5 10 15 GlnThr Thr Trp Lys Gly Leu Trp Met Ser Cys Val Val Gln 20 25 30 471 30 PRTHomo sapien 471 Pro Met Trp Gln Val Thr Ala Phe Leu Asp His Asn Ile ValThr Ala 1 5 10 15 Gln Thr Thr Trp Lys Gly Leu Trp Met Ser Cys Val ValGln 20 25 30 472 30 PRT Mus musculus 472 Pro Met Trp Lys Val Thr Ala PheIle Gly Asn Ser Ile Val Val Ala 1 5 10 15 Gln Met Val Trp Glu Gly LeuTrp Met Ser Cys Val Val Gln 20 25 30 473 30 PRT Mus musculus 473 Pro GlnTrp Gln Met Ser Ser Tyr Ala Gly Asp Asn Ile Ile Thr Ala 1 5 10 15 GlnAla Met Tyr Lys Gly Leu Trp Met Glu Cys Val Thr Gln 20 25 30 474 30 PRTMus musculus 474 Pro Gln Trp Arg Val Ser Ala Phe Ile Glu Ser Asn Ile ValVal Phe 1 5 10 15 Glu Asn Arg Trp Glu Gly Leu Trp Met Asn Cys Met ArgHis 20 25 30 475 10 PRT Artificial Sequence Representative linearmodulating agent based on claudin-1 cell adhesion recognition sequenceused in Example 5 to demonstrate electrical resistance across cellmonolayer 475 Trp Lys Ile Tyr Ser Tyr Ala Gly Asp Asn 1 5 10 476 9 PRTArtificial Sequence Representative linear modulating agent based onclaudin-1 cell adhesion recognition sequence 476 Trp Lys Ile Tyr Ser TyrAla Gly Asn 1 5 477 9 PRT Artificial Sequence Representative celladhesion recognition sequence which is bound by JAM, a member of theimmunoglobulin supergene family 477 Ser Phe Thr Ile Asp Pro Lys Ser Gly1 5 478 10 PRT Artificial Sequence Representative occuldin cell adhesionrecognition sequence 478 Gln Tyr Leu Tyr His Tyr Cys Val Val Asp 1 5 10479 9 PRT Artificial Sequence Representative OB-cadherin cell adhesionrecognition sequence 479 Ile Phe Val Ile Asp Asp Lys Ser Gly 1 5 480 5PRT Artificial Sequence Representative linear modulating agent based onclaudin-1 cell adhesion recognition sequence 480 Arg Ile Tyr Ser Tyr 1 5481 6 PRT Artificial Sequence Representative linear modulating agentbased on claudin-1 cell adhesion recognition sequence 481 Arg Ile TyrSer Tyr Ala 1 5 482 7 PRT Artificial Sequence Representative linearmodulating agent based on claudin-1 cell adhesion recognition sequence482 Arg Ile Tyr Ser Tyr Ala Gly 1 5 483 6 PRT Artificial SequenceRepresentative linear modulating agent based on claudin-1 cell adhesionrecognition sequence 483 Trp Arg Ile Tyr Ser Tyr 1 5 484 7 PRTArtificial Sequence Representative linear modulating agent based onclaudin-1 cell adhesion recognition sequence 484 Trp Arg Ile Tyr Ser TyrAla 1 5 485 8 PRT Artificial Sequence Representative linear modulatingagent based on claudin-1 cell adhesion recognition sequence 485 Trp ArgIle Tyr Ser Tyr Ala Gly 1 5 486 9 PRT Artificial Sequence Representativelinear modulating agent based on claudin-1 cell adhesion recognitionsequence 486 Trp Arg Ile Tyr Ser Tyr Ala Gln Asn 1 5 487 6 PRTArtificial Sequence Representative cyclic modulating agent based onclaudin-1 cell adhesion recognition sequence 487 Cys Arg Ile Tyr Ser Tyr1 5 488 8 PRT Artificial Sequence Representative cyclic modulating agentbased on claudin-1 cell adhesion recognition sequence 488 Cys Arg IleTyr Ser Tyr Ala Cys 1 5 489 9 PRT Artificial Sequence Representativecyclic modulating agent based on claudin-1 cell adhesion recognitionsequence 489 Cys Arg Ile Tyr Ser Tyr Ala Gly Cys 1 5 490 8 PRTArtificial Sequence Representative cyclic modulating agent based onclaudin-1 cell adhesion recognition sequence 490 Cys Trp Arg Ile Tyr SerTyr Cys 1 5 491 9 PRT Artificial Sequence Representative cyclicmodulating agent based on claudin-1 cell adhesion recognition sequence491 Cys Trp Arg Ile Tyr Ser Tyr Ala Cys 1 5 492 10 PRT ArtificialSequence Representative cyclic modulating agent based on claudin-1 celladhesion recognition sequence 492 Cys Trp Arg Ile Tyr Ser Tyr Ala GlyCys 1 5 10 493 7 PRT Artificial Sequence Representative cyclicmodulating agent based on claudin-1 cell adhesion recognition sequence493 Lys Arg Ile Tyr Ser Tyr Asp 1 5 494 8 PRT Artificial SequenceRepresentative cyclic modulating agent based on claudin-1 cell adhesionrecognition sequence 494 Lys Arg Ile Tyr Ser Tyr Ala Asp 1 5 495 9 PRTArtificial Sequence Representative cyclic modulating agent based onclaudin-1 cell adhesion recognition sequence 495 Lys Arg Ile Tyr Ser TyrAla Gly Asp 1 5 496 8 PRT Artificial Sequence Representative cyclicmodulating agent based on claudin-1 cell adhesion recognition sequence496 Lys Trp Arg Ile Tyr Ser Tyr Asp 1 5 497 9 PRT Artificial SequenceRepresentative cyclic modulating agent based on claudin-1 cell adhesionrecognition sequence 497 Lys Trp Arg Ile Tyr Ser Tyr Ala Asp 1 5 498 10PRT Artificial Sequence Representative cyclic modulating agent based onclaudin-1 cell adhesion recognition sequence 498 Lys Trp Arg Ile Tyr SerTyr Ala Gly Asp 1 5 10 499 7 PRT Artificial Sequence Representativecyclic modulating agent based on claudin-1 cell adhesion recognitionsequence 499 Lys Arg Ile Tyr Ser Tyr Glu 1 5 500 8 PRT ArtificialSequence Representative cyclic modulating agent based on claudin-1 celladhesion recognition sequence 500 Lys Arg Ile Tyr Ser Tyr Ala Glu 1 5501 9 PRT Artificial Sequence Representative cyclic modulating agentbased on claudin-1 cell adhesion recognition sequence 501 Lys Arg IleTyr Ser Tyr Ala Gly Glu 1 5 502 8 PRT Artificial Sequence Representativecyclic modulating agent based on claudin-1 cell adhesion recognitionsequence 502 Lys Trp Arg Ile Tyr Ser Tyr Glu 1 5 503 9 PRT ArtificialSequence Representative cyclic modulating agent based on claudin-1 celladhesion recognition sequence 503 Lys Trp Arg Ile Tyr Ser Tyr Ala Glu 15 504 10 PRT Artificial Sequence Representative cyclic modulating agentbased on claudin-1 cell adhesion recognition sequence 504 Lys Trp ArgIle Tyr Ser Tyr Ala Gly Glu 1 5 10 505 7 PRT Artificial SequenceRepresentative cyclic modulating agent based on claudin-1 cell adhesionrecognition sequence 505 Asp Arg Ile Tyr Ser Tyr Lys 1 5 506 8 PRTArtificial Sequence Representative cyclic modulating agent based onclaudin-1 cell adhesion recognition sequence 506 Asp Arg Ile Tyr Ser TyrAla Lys 1 5 507 9 PRT Artificial Sequence Representative cyclicmodulating agent based on claudin-1 cell adhesion recognition sequence507 Asp Arg Ile Tyr Ser Tyr Ala Gly Lys 1 5 508 8 PRT ArtificialSequence Representative cyclic modulating agent based on claudin-1 celladhesion recognition sequence 508 Asp Trp Arg Ile Tyr Ser Tyr Lys 1 5509 9 PRT Artificial Sequence Representative cyclic modulating agentbased on claudin-1 cell adhesion recognition sequence 509 Asp Trp ArgIle Tyr Ser Tyr Ala Lys 1 5 510 10 PRT Artificial SequenceRepresentative cyclic modulating agent based on claudin-1 cell adhesionrecognition sequence 510 Asp Trp Arg Ile Tyr Ser Tyr Ala Gly Lys 1 5 10511 7 PRT Artificial Sequence Representative cyclic modulating agentbased on claudin-1 cell adhesion recognition sequence 511 Glu Arg IleTyr Ser Tyr Lys 1 5 512 8 PRT Artificial Sequence Representative cyclicmodulating agent based on claudin-1 cell adhesion recognition sequence512 Glu Arg Ile Tyr Ser Tyr Ala Lys 1 5 513 9 PRT Artificial SequenceRepresentative cyclic modulating agent based on claudin-1 cell adhesionrecognition sequence 513 Glu Arg Ile Tyr Ser Tyr Ala Gly Lys 1 5 514 8PRT Artificial Sequence Representative cyclic modulating agent based onclaudin-1 cell adhesion recognition sequence 514 Glu Trp Arg Ile Tyr SerTyr Lys 1 5 515 9 PRT Artificial Sequence Representative cyclicmodulating agent based on claudin-1 cell adhesion recognition sequence515 Glu Trp Arg Ile Tyr Ser Tyr Ala Lys 1 5 516 10 PRT ArtificialSequence Representative cyclic modulating agent based on claudin-1 celladhesion recognition sequence 516 Glu Trp Arg Ile Tyr Ser Tyr Ala GlyLys 1 5 10 517 6 PRT Artificial Sequence Representative cyclicmodulating agent based on claudin-1 cell adhesion recognition sequence517 Trp Arg Ile Tyr Ser Tyr 1 5 518 7 PRT Artificial SequenceRepresentative cyclic modulating agent based on claudin-1 cell adhesionrecognition sequence 518 Trp Arg Ile Tyr Ser Tyr Ala 1 5 519 8 PRTArtificial Sequence Representative cyclic modulating agent based onclaudin-1 cell adhesion recognition sequence 519 Trp Arg Ile Tyr Ser TyrAla Gly 1 5 520 41 PRT Mus musculus 520 Asp Tyr Trp Lys Val Ser Thr IleAsp Gly Thr Val Ile Thr Thr Ala 1 5 10 15 Thr Tyr Phe Ala Asn Leu TrpLys Ile Cys Val Thr Asp Ser Thr Gly 20 25 30 Val Ala Asn Cys Lys Glu PhePro Ser 35 40 521 41 PRT Homo sapien 521 Asp Tyr Trp Lys Val Ser Thr IleAsp Gly Thr Val Ile Thr Thr Ala 1 5 10 15 Thr Tyr Trp Ala Asn Leu TrpLys Ala Cys Val Thr Asp Ser Thr Gly 20 25 30 Val Ser Asn Cys Lys Asp PhePro Ser 35 40 522 43 PRT Mus musculus 522 Asn Asp Trp Val Val Thr CysSer Tyr Thr Ile Pro Thr Cys Arg Lys 1 5 10 15 Met Asp Glu Leu Gly SerLys Gly Leu Trp Ala Asp Cys Val Met Ala 20 25 30 Thr Gly Leu Tyr His CysLys Pro Leu Val Asp 35 40 523 43 PRT Homo sapien 523 Asn Asp Trp Val ValThr Cys Gly Tyr Thr Ile Pro Thr Cys Arg Lys 1 5 10 15 Leu Asp Glu LeuGly Ser Lys Gly Leu Trp Ala Asp Cys Val Met Ala 20 25 30 Thr Gly Leu TyrHis Cys Lys Pro Leu Val Asp 35 40 524 41 PRT Mus musculus 524 Pro ValTrp Arg Val Thr Phe Pro Asp Asp Glu Thr Asp Pro Asp Ala 1 5 10 15 ThrIle Trp Glu Gly Leu Trp His Ile Cys Gln Val Arg Glu Asn Arg 20 25 30 TrpIle Gln Cys Thr Leu Tyr Asp Thr 35 40 525 42 PRT Mus musculus 525 ProHis Trp Arg Arg Thr Ala His Val Gly Thr Asn Ile Leu Thr Ala 1 5 10 15Val Ser Tyr Leu Lys Gly Leu Trp Met Glu Cys Val Trp His Ser Thr 20 25 30Gly Ile Tyr Gln Cys Gln Ile Tyr Arg Ser 35 40 526 42 PRT Homo sapien 526Pro His Trp Arg Arg Thr Ala His Val Gly Thr Asn Ile Leu Thr Ala 1 5 1015 Val Ser Tyr Leu Lys Gly Leu Trp Met Glu Cys Val Trp His Ser Thr 20 2530 Gly Ile Tyr Gln Cys Gln Ile Tyr Arg Ser 35 40 527 41 PRT Homo sapien527 Ser Tyr Trp Arg Val Ser Thr Val His Gly Asn Val Ile Thr Thr Asn 1 510 15 Thr Ile Phe Glu Asn Leu Trp Phe Ser Cys Ala Thr Asp Ser Leu Gly 2025 30 Val Tyr Asn Cys Trp Glu Phe Pro Ser 35 40 528 43 PRT Bos taurus528 Thr Trp Thr Asp Cys Trp Met Val Asn Ala Asp Asp Ser Leu Glu Val 1 510 15 Ser Thr Lys Cys Arg Gly Leu Trp Trp Glu Cys Val Thr Asn Ala Phe 2025 30 Asp Gly Ile Arg Thr Cys Asp Glu Tyr Asp Ser 35 40 529 42 PRT Homosapien 529 Pro Gln Trp Arg Ile Tyr Ser Tyr Ala Gly Asp Asn Ile Val ThrAla 1 5 10 15 Gln Ala Met Tyr Glu Gly Leu Trp Met Ser Cys Val Ser GlnSer Thr 20 25 30 Gly Gln Val Gln Cys Lys Val Phe Asp Ser 35 40 530 42PRT Homo sapien 530 Pro Met Trp Lys Val Thr Ala Phe Ile Gly Asn Ser IleVal Val Ala 1 5 10 15 Gln Val Val Trp Glu Gly Leu Trp Met Ser Cys ValVal Gln Ser Thr 20 25 30 Gly Gln Met Gln Cys Lys Val Tyr Asp Ser 35 40531 42 PRT Homo sapien and Mus musculus 531 Pro Leu Trp Lys Val Thr AlaPhe Ile Gly Asn Ser Ile Val Val Ala 1 5 10 15 Gln Val Val Trp Glu GlyLeu Trp Met Ser Cys Val Val Gln Ser Thr 20 25 30 Gly Gln Met Gln Cys LysVal Tyr Asp Ser 35 40

What is claimed is:
 1. A method for inhibiting claudin-mediated celladhesion, comprising contacting a claudin-expressing cell with a celladhesion modulating agent that: (a) comprises an acetylated claudin CARsequence selected from the group consisting of WKIYSYAG (SEQ ID NO: 34),WRIYSYAG (SEQ ID NO: 485), WRTSSYVG (SEQ ID NO: 42), WRVSAFIG (SEQ IDNO: 58), WRVTAFIG (SEQ ID NO: 50), WQVTAFLD (SEQ ID NO: 277), WQMSSYAG(SEQ ID NO: 394), WRVSAFIE (SEQ ID NO: 450), and WKVTAFIG (SEQ ID NO:335), said claudin CAR sequence being present in a naturally occurringclaudin; and (b) consists of no more than 50 consecutive amino acidresidues present within the claudin.
 2. A method according to claim 1,wherein the modulating agent is a peptide ranging in size from 8 to 50amino acid residues.
 3. A method according to claim 1, wherein themodulating agent is a peptide ranging in size from 8 to 16 amino acidresidues.
 4. A method according to claim 1, wherein the modulating agentis linked to a detectable marker.
 5. A method according to claim 1,wherein the modulating agent is linked to a support material.
 6. Amethod according to claim 5, wherein the support material is a polymericmatrix.
 7. A method according to claim 5, wherein the support materialis selected from the group consisting of plastic dishes, plastic tubes,sutures, membranes, ultra thin films, bioreactors and microparticles. 8.A method according to claim 1, wherein the modulating agent is presentwithin a composition comprising an acceptable carrier.